. 2019 May 31;9:8120. doi: 10.1038/s41598-019-44542-3

Table 1.

PfpTKL domain/motif annotation results.

Domain/motif name	PfpTKL region (AA)	Data from the literature	PfpTKL annotation data (this study)
Kinase domain (KD)	1080–1483 (Fig. 1a see Supplementary Table S2 for details)	Kinase domains are composed of 12 subdomains²³, six of which are critical for kinase activity²⁵. PfpTKL lacks some canonical KD subdomains⁴¹. PbpTKL is a pseudokinase¹⁹.	PfpTKL KD (Fig. 1a): —belongs to the tyrosine kinase-like (TKL) family⁴² (Supplementary Fig. S1). —lacks the following subdomains: -I (glycine triad) -II (ATP-binding lysine) although there might be an alignment offset because the PfpTKL KD N-terminal lobe is longer than in reference kinases -III (αC helix glutamate can interact with subdomain II lysine) —includes some specificities: -VIB aspartate (ATP γ-phosphate transfer catalyzer) is mutated to asparagine -a ~50-residue insertion in its activation loop (shown in red in Fig. 1a).
Bruton’s tyrosine kinase-like (BTK-like) domain	830–905 (Fig. 1b)	BTK is named after Dr Ogden Carr Bruton, who discovered X-linked agammaglobulinemia (XLA) in boys in 1952. This cytoplasmic tyrosine kinase (mostly in B lymphocytes) needs to bind lipids via its PH-TH module to adopt an active conformation¹⁰⁴. BTK is a promising target in tumors¹⁰⁵.	BTK-like domain: —was predicted by most of the prediction tools we used. —alignment with HsPKA and HsBTK N-terminal lobe sequences shows the conservation of canonical KD subdomains II (this lysine is shaded in red in Fig. 1b), IV (highlighted in gray) and V. —tertiary structure prediction superimposes well with HsBTK N-terminal lobe (Supplementary Fig. S3).
Membrane occupation recognition nexus (MORN) domain	MORN1: 19–40 MORN2: 43–63 (Fig. 2a)	First identified in Junctophilins¹⁰⁶, MORN motifs have been shown to bind: -lipids and regulate kinase activity in plant PIPKs^107,108 -the cytoskeleton as in TgMORN1¹⁰⁹ -membranes and is required for the localization of retinophilin in the Drosophila melanogaster rhabdomere¹¹⁰	There is no signal peptide at the N-terminus of PfpTKL. MORN1 follows the MORN motif signature: a 14–16 residue core domain starting with Y/FxG and ending with GxG (Supplementary Fig. S4). MORN2 does not follow a canonical MORN signature but contains a putative N-myristoylation site that is conserved across all Plasmodium species (Fig. 2a).
RVxF motifs	RVxF1: 482–486 RVxF2: 1219–1223 (Fig. 2b)	RVxF motifs: - so-called because of their binding signature¹¹¹ - are the most frequent binding motifs used by PP1 partners to interact with PP1c¹¹² - can be predicted with consensus sequences (Fig. 2b)	RVxF1 follows the Meiselbach consensus sequence⁸⁷. It is located in a low-complexity region, which may increase its interaction potential with PP1c⁵⁰. It is conserved among PfpTKL homologs in P. praefalciparum, P. reichenowi, P. billcollinsi and P. blacklocki (Fig. 3c). RVXF2 follows the Wakula consensus sequence⁸⁶. It is located in a basic charged region, which could reinforce its binding potential⁷⁵. It is conserved across all PfpTKL homologs in the genus Plasmodium (Figs 2b and 3c).
Sterile alpha motif (SAM)	299–364 (Fig. 2d)	SAMs belong to the alpha protein family (289 folds – SCOP database¹¹³). They typically fold into 4–5 orthogonal α helices. They are involved in interactions with protein or RNA^71,72. In the Plasmodium proteome: - there are four SAMs (PfpTKL, PfTKL1, PfTKL3, PF3D7_0926000)⁶⁶ - there are two SAM-like domains involved in interactions with RNA (prototypical HTH motifs) in Pfg27^114,115.	The PfpTKL SAM domain is predicted to fold classically (Fig. 2c). The PfpTKL SAM core domain includes all SAM canonical residues (Fig. 2d). The PfpTKL SAM domain is close to SAMs involved in protein–protein interactions (Supplementary Fig. S5). This phylogenetic analysis includes Plasmodium SAM and SAM-like sequences as well as reference SAM and SAM-like sequences across the tree of life.

Domain/motif name

PfpTKL region (AA)

Data from the literature

PfpTKL annotation data (this study)

Kinase domain (KD)

1080–1483

(Fig. 1a see Supplementary Table S2 for details)

Kinase domains are composed of 12 subdomains²³, six of which are critical for kinase activity²⁵.

PfpTKL lacks some canonical KD subdomains⁴¹.

PbpTKL is a pseudokinase¹⁹.

PfpTKL KD (Fig. 1a):

—belongs to the tyrosine kinase-like (TKL) family⁴² (Supplementary Fig. S1).

—lacks the following subdomains:

-I (glycine triad)

-II (ATP-binding lysine) although there might be an alignment offset because the PfpTKL KD N-terminal lobe is longer than in reference kinases

-III (αC helix glutamate can interact with subdomain II lysine)

—includes some specificities:

-VIB aspartate (ATP γ-phosphate transfer catalyzer) is mutated to asparagine

-a ~50-residue insertion in its activation loop (shown in red in Fig. 1a).

Bruton’s tyrosine kinase-like (BTK-like) domain

830–905

(Fig. 1b)

BTK is named after Dr Ogden Carr Bruton, who discovered X-linked agammaglobulinemia (XLA) in boys in 1952. This cytoplasmic tyrosine kinase (mostly in B lymphocytes) needs to bind lipids via its PH-TH module to adopt an active conformation¹⁰⁴. BTK is a promising target in tumors¹⁰⁵.

BTK-like domain:

—was predicted by most of the prediction tools we used.

—alignment with HsPKA and HsBTK N-terminal lobe sequences shows the conservation of canonical KD subdomains II (this lysine is shaded in red in Fig. 1b), IV (highlighted in gray) and V.

—tertiary structure prediction superimposes well with HsBTK N-terminal lobe (Supplementary Fig. S3).

Membrane occupation recognition nexus (MORN) domain

MORN1:

19–40

MORN2:

43–63

(Fig. 2a)

First identified in Junctophilins¹⁰⁶, MORN motifs have been shown to bind:

-lipids and regulate kinase activity in plant PIPKs^107,108

-the cytoskeleton as in TgMORN1¹⁰⁹

-membranes and is required for the localization of retinophilin in the Drosophila melanogaster rhabdomere¹¹⁰

There is no signal peptide at the N-terminus of PfpTKL.

MORN1 follows the MORN motif signature: a 14–16 residue core domain starting with Y/FxG and ending with GxG (Supplementary Fig. S4).

MORN2 does not follow a canonical MORN signature but contains a putative N-myristoylation site that is conserved across all Plasmodium species (Fig. 2a).

RVxF motifs

RVxF1:

482–486

RVxF2:

1219–1223

(Fig. 2b)

RVxF motifs:

- so-called because of their binding signature¹¹¹

- are the most frequent binding motifs used by PP1 partners to interact with PP1c¹¹²

- can be predicted with consensus sequences (Fig. 2b)

RVxF1 follows the Meiselbach consensus sequence⁸⁷. It is located in a low-complexity region, which may increase its interaction potential with PP1c⁵⁰. It is conserved among PfpTKL homologs in P. praefalciparum, P. reichenowi, P. billcollinsi and P. blacklocki (Fig. 3c).

RVXF2 follows the Wakula consensus sequence⁸⁶. It is located in a basic charged region, which could reinforce its binding potential⁷⁵. It is conserved across all PfpTKL homologs in the genus Plasmodium (Figs 2b and 3c).

Sterile alpha motif (SAM)

299–364

(Fig. 2d)

SAMs belong to the alpha protein family (289 folds – SCOP database¹¹³). They typically fold into 4–5 orthogonal α helices. They are involved in interactions with protein or RNA^71,72.

In the Plasmodium proteome:

- there are four SAMs (PfpTKL, PfTKL1, PfTKL3, PF3D7_0926000)⁶⁶

- there are two SAM-like domains involved in interactions with RNA (prototypical HTH motifs) in Pfg27^114,115.

The PfpTKL SAM domain is predicted to fold classically (Fig. 2c).

The PfpTKL SAM core domain includes all SAM canonical residues (Fig. 2d).

The PfpTKL SAM domain is close to SAMs involved in protein–protein interactions (Supplementary Fig. S5). This phylogenetic analysis includes Plasmodium SAM and SAM-like sequences as well as reference SAM and SAM-like sequences across the tree of life.