Phosphorylation of spore coat proteins by a family of atypical protein kinases - PubMed (original) (raw)

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. 2016 Jun 21;113(25):E3482-91.

doi: 10.1073/pnas.1605917113. Epub 2016 May 16.

Anju Sreelatha 2, Eric S Durrant 1, Javier Lopez-Garrido 3, Anna Muszewska 4, Małgorzata Dudkiewicz 5, Marcin Grynberg 6, Samantha Yee 2, Kit Pogliano 3, Diana R Tomchick 7, Krzysztof Pawłowski 5, Jack E Dixon 8, Vincent S Tagliabracci 9

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Phosphorylation of spore coat proteins by a family of atypical protein kinases

Kim B Nguyen et al. Proc Natl Acad Sci U S A. 2016.

Abstract

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

Keywords: CotB; CotG; CotH; kinase; phosphorylation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

CotH proteins share sequence similarity with known protein kinases. (A) Phylogenetic tree depicting the eukaryotic secretory pathway kinases and related prokaryotic proteins: Fam20A/B, Fam198A/B, four-jointed box 1 (Fjx1), and HipA. Sequence logos were created by WebLogo, using MUSCLE multiple sequence alignment of 165 CotH homologs depicting the loop between strands β1 and β2 (B, Gly-rich loop) and the active site residues (C).

Fig. S1.

Fig. S1.

CotH proteins share sequence similarity with protein kinases. CLANS clustering analysis is represented graphically as the network of BLAST-derived sequence similarities (edges) between representatives of known kinase families (dots). All PKL clan families are included, including those clan families predicted to have a PKL fold, such as the Fam69 family. Magenta, CotH; red, Fam20; pink, Fam198; brown, HipA; dark blue, PI3_PI4K; light green, actin-fragmin kinases; light blue, CotS; cyan, Fam69; yellow, pkinase (typical eukaryotic Ser/Thr and Tyr kinases).

Fig. S2.

Fig. S2.

Multiple sequence alignment of selected CotH proteins and other kinases. The alignment was generated with PROMALS3D (77) and includes selected CotH homologs (representatives of clusters seen in Fig. 7), with additional related kinases [Fam20C, phosphatidylinositol 4-kinase IIα (PI4K), HipA, CtkA, PKA, and CotS]. National Center for Biotechnology Information GeneInfo identifier (gi) numbers are shown.

Fig. 2.

Fig. 2.

B. subtilis and B. cereus CotH are active protein kinases. (A) Incorporation of 32P from [γ-32P]ATP into B. subtilis and B. cereus wild-type (WT) CotH or the indicated mutants. Reaction products were separated by SDS/PAGE and stained with Coomassie blue. The incorporated radioactivity was visualized by autoradiography. Incorporation of 32P from [γ-32P]ATP into MyeBP, H2A, and OPN by B. subtilis wild-type CotH or the indicated mutants (B) and B. cereus wild-type CotH or the indicated mutants (C). Reaction products were analyzed as in A. (D) Incorporation of 32P from [γ-32P]ATP into a peptide substrate, CotG(88–100), which is derived from the CotH substrate CotG (described in Fig. 6) by B. subtilis CotH. The reactions were carried out in the presence of 5 mM MgCl2, MnCl2, CaCl2, CoCl2, FeCl2, or ZnCl2, and the reaction products were spotted on P81 phosphocellulose filter papers and terminated by immersion in H3PO4. Filter papers were washed, and incorporated radioactivity was quantified by scintillation counting.

Fig. 3.

Fig. 3.

Crystal structure of B. cereus CotH reveals an atypical PKL fold. (A) Ribbon representation of B. cereus CotH. The N- and C-lobes are shown in magenta and teal, respectively. The α2 helix (PKA αC equivalent) is highlighted in orange. (B) Amino acid sequence of B. cereus CotH depicting the secondary structural elements, color-coded as in A. The 310 helices (η) are also shown.

Fig. S3.

Fig. S3.

Structural comparison of B. cereus CotH with PKA and selected atypical kinases. The structures of B. cereus CotH, PKA (PDB ID code 1ATP), Caenorhabditis elegans Fam20C (PDB ID code 4KQB), CtkA (PDB ID code 3AKJ), HipA (PDB ID code 3FBR), and PI4K IIα (PDB ID code 4HND) are shown. The α-helices are colored in red, and the β-strands are colored in yellow.

Fig. 4.

Fig. 4.

Mg2+/AMP-bound structure of B. cereus CotH reveals a unique mode of nucleotide binding and highlights residues involved in catalysis. (A) Surface representations illustrating the electrostatic potential of B. cereus CotH depicting AMP (yellow sticks) and Mg2+ (cyan spheres) bound in a cleft between the N- and C-lobes of the kinase. The electrostatic potentials were calculated with the APBS2.1 plug-in of PyMOL (65). The gradients of electrostatic potentials shown ranged from ≥−5 kbT/ec (red) to ≤+5 kbT/ec (blue), where kb is Boltzmann’s constant, T is temperature in degrees Kelvin, and ec is the charge of an electron. (B) Expanded image of the nucleotide-binding pocket showing the detailed molecular interactions important for nucleotide binding and catalysis. The AMP molecule and the two Mg2+ ions are shown as green sticks and cyan spheres, respectively. The salt bridge and hydrogen bond interactions are shown as dashed lines. (C) Kinetic analysis depicting the concentration dependence of Mn2+/ATP on the rate of phosphate incorporation into CotG(88–100) by B. subtilis CotH. (Inset) K_m for Mn2+/ATP is indicated. Reaction products were analyzed as in Fig. 2_D. (D) Kinase activity of B. subtilis CotH and active site mutants was analyzed as in Fig. 2_D_. Activity is expressed relative to activity of the wild-type enzyme. The amino acids in brackets indicate the corresponding residues in B. cereus CotH.

Fig. S4.

Fig. S4.

Comparison of the active sites in the apo- and AMP/Mg2+ structures. Expanded images of the nucleotide-binding pocket in the apo structure (A) and the AMP/Mg2+ structure (B) are shown. The AMP molecule and the two Mg2+ ions are shown as green sticks and cyan spheres, respectively. Interactions are shown as dashed lines.

Fig. 5.

Fig. 5.

CotH kinase activity is required for the proper germination of B. subtilis spores. (A) Germination efficiency was analyzed in spores derived from the strains displayed in Table S2. Germination was induced by L-alanine and measured as the percentage of loss of optical density at 580 nm. (B) Protein immunoblotting of mother cell extracts depicting the time-dependent expression of V5-tagged wild-type CotH and the D251A mutant following induction of sporulation. α-SigA immunoblots are shown as a loading control. (C, Upper) Protein immunoblotting of spore coat extracts depicting the incorporation of V5-tagged CotH into the spore coat. (C, Lower) Ponceau S-stained membrane is shown as a loading control.

Fig. 6.

Fig. 6.

B. subtilis CotH phosphorylates the spore coat proteins CotB and CotG. (A) B. subtilis cotH gene forms a cluster with two genes that encode spore coat proteins cotB and cotG. (B) Schematic representation of B. subtilis CotB depicting the Ser/Arg/Lys-rich C-terminal region. (C) Time-dependent incorporation of 32P from [γ-32P]ATP into 6× His-tagged CotB by B. subtilis CotH. Reaction products were separated by SDS/PAGE and visualized by Coomassie staining (Upper), and radioactivity was detected by autoradiography (Lower). (D) Schematic representation of B. subtilis CotG depicting the nine tandem repeats in the protein. Representative tandem mass spectroscopy (MS/MS) fragmentation spectra depicting Ser4 (E) and Ser7 (F) phosphorylation of CotG(88–100) by CotH is shown. (G, Upper) Protein immunoblotting of B. subtilis spore coat extracts using a phosphospecific PKC substrate antibody depicting the phosphorylation of spore coat proteins in the different strains. (G, Lower) Ponceau S-stained membrane is shown as a loading control. The asterisks depict proteins that we infer are CotB and CotG based on their molecular masses.

Fig. 7.

Fig. 7.

Taxonomy, sporulation, and pathogenicity of CotH-possessing organisms. (A) CLANS clustering analysis of CotH homologs represented graphically by the network of BLAST-derived sequence similarities (edges) between representative CotH proteins (dots). Clusters are colored by taxonomy. Roman numerals denote sequence clusters (representatives in the alignment are shown in Fig. S2). CLANS clustering analysis of CotH homologs is colored by ability to sporulate (B) or by pathogenicity (C).

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