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Structural and functional diversity of the microbial kinome - PubMed

Structural and functional diversity of the microbial kinome

Natarajan Kannan et al. PLoS Biol. 2007 Mar.

Abstract

The eukaryotic protein kinase (ePK) domain mediates the majority of signaling and coordination of complex events in eukaryotes. By contrast, most bacterial signaling is thought to occur through structurally unrelated histidine kinases, though some ePK-like kinases (ELKs) and small molecule kinases are known in bacteria. Our analysis of the Global Ocean Sampling (GOS) dataset reveals that ELKs are as prevalent as histidine kinases and may play an equally important role in prokaryotic behavior. By combining GOS and public databases, we show that the ePK is just one subset of a diverse superfamily of enzymes built on a common protein kinase-like (PKL) fold. We explored this huge phylogenetic and functional space to cast light on the ancient evolution of this superfamily, its mechanistic core, and the structural basis for its observed diversity. We cataloged 27,677 ePKs and 18,699 ELKs, and classified them into 20 highly distinct families whose known members suggest regulatory functions. GOS data more than tripled the count of ELK sequences and enabled the discovery of novel families and classification and analysis of all ELKs. Comparison between and within families revealed ten key residues that are highly conserved across families. However, all but one of the ten residues has been eliminated in one family or another, indicating great functional plasticity. We show that loss of a catalytic lysine in two families is compensated by distinct mechanisms both involving other key motifs. This diverse superfamily serves as a model for further structural and functional analysis of enzyme evolution.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Sequence and Structure Based Clustering of PKL Families

Despite minimal sequence similarity, relationships between families can be estimated by profile–profile matching and alignments restricted to conserved motifs. Three main clusters of families are seen (shaded ovals): CAK, ePK, and KdoK. Four more families (towards bottom) are distantly related to these clusters, while three more (PI3K, AlphaK, IDHK, at bottom) have no sequence similarity outside a subset of key motifs. The area of each sphere represents the family size within GOS data.

Figure 2
Figure 2. The Conserved Core and Variable Regions of the Catalytic Domain

The conserved core in three distinct families, namely ePK (PKA [52]), Rio (A. fulgidis Rio2 [14]), and CAK (APH(3′)-IIIa [12]). The conserved regions are shown in ribbon representation and the variable regions in surface representation. The illustrations were created in PyMOL (

http://www.pymol.org

). Some highly conserved residues (see Figure 3) and their associated interactions are shown.

Figure 3
Figure 3. Conservation of Secondary Structure, Key Motifs, and Residues between Families

The ePK secondary structure is shown with standard annotations of subdomains [53] and structural elements. Subdomains I–IX are generally conserved in all PKLs. Key residues are bolded and numbered; dashed lines point to positions within secondary structure elements. The table below shows the conservation (% identity) of the ten key residues, showing their broad conservation across families, but the successful replacement of almost all of them in at least one family. Parentheses indicate changes to another conserved residue and dashes indicate unconserved positions. Key residues are numbered based on their position in PKA: G52, K72, E91, P104 (VPKA), H158, H164 (YPKA), D166, N171, D184, and D220. More detailed figures are shown in Dataset S3.

Figure 4
Figure 4. Sequence Logos Depicting Conservation of Core Motifs and Neighboring Sequences across Most Kinase Families and Selected CAK Subfamilies

Motifs are GxGxxGxxxx, VAIK, E, LxxLH, xxHxDxxxxNxx, xxDFGxx, and Dxx. The size of the letters corresponds to their information content [54]. Families with less than 100 members (BLRK, GLK) are omitted. The diverse CAK family is represented by four distinct subfamilies: APH contains many aminoglycoside resistance kinases and ChoK includes most ChoKs, while FadE and chloro are less well described. For the HRK family, the first two motif logos omit the viral subfamily that lacks these motifs.

Figure 5
Figure 5. Mechanistic Diversity of the ATP-Binding Pocket.

(A) PKA showing structural interactions associated with K72 in active ATP-bound state. The salt bridge interaction between K72 and E91 is shown by dotted lines. (B) Structural interactions associated with Arg111ChoK in ChoK. (C) Conformational changes associated with Arg52Erk2 in the Erk2 mutant structure. Here, the arginine does not form a salt bridge interaction with Glu69Erk2 (E91), but moves closer towards Glu69Erk2 upon ATP binding. (D) Inactive state of Wnk1: K72 is shifted over to the G-loop (K233Wnk1) and E91 (Glu268Wnk1) hydrogen bonds to a conserved Arg (R348Wnk1 within the HRD motif) in the catalytic loop. (A–D) Residues conserved across all the major families are colored in magenta, while family-specific residues are colored in gold. Hydrogen bonds are indicated in dotted lines.

Figure 6
Figure 6. ePK-Specific Motifs and Interactions in the Substrate-Binding Region

(A) The ePK-specific activation loop and G-helix are shown in PKA (PKA [52]). The corresponding regions are shown in Rio (A. fulgidis Rio2 [14]). The activation loop and G-helix are colored in red, and the core-conserved residues are shown in stick representation. (B) The three ePK-specific motifs in the C-terminal substrate-binding lobe and their structural interactions are shown. Hydrogen bonds are indicated by dotted lines. The conserved buried water is shown in CPK representation.

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