Natarajan Kannan

Structural and functional diversity of the microbial kinome.

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.

The hallmark of AGC kinase functional divergence is its C-terminal tail, a cis-acting regulatory module

The catalytic activities of eukaryotic protein kinases (EPKs) are regulated by movement of the C-helix, movement of the N and C lobes upon ATP binding, and movement of the activation loop upon phosphorylation. Statistical analysis of the selective constraints associated with AGC kinase functional divergence reveals conserved interactions between these regulatory regions and three regions of the C-terminal tail (C-tail): the N-lobe tether (NLT), the active-site tether (AST), and the C-lobe tether (CLT). The NLT serves as a docking site for an upstream kinase PDK1 and, upon activation, positions the C-helix within the ATP binding pocket. The AST directly interacts with the ATP binding pocket, and the CLT interacts with the interlobe linker and the αC–β4 loop, which appears to serve as a hinge for C-helix movement. The C-tail is a hallmark of AGC functional divergence inasmuch as most of the conserved core residues that distinguish AGC kinases from other EPKs are associated with the NLT, AST, or CLT. Moreover, several AGC catalytic core conserved residues that interact with the C-tail strikingly diverge from the canonical residues observed at corresponding positions in nearly all other EPKs, suggesting that the catalytic core may have coevolved with the C-tail in AGC kinases. These observations, along with the fact that the C-tail is needed for catalytic activity suggests that the C-tail is a cis-acting regulatory module that can also serve as a regulatory “handle,” to which trans-acting cellular components can bind to modulate activity.

PROTEIN STRUCTURE: INSIGHTS FROM GRAPH THEORY

The sequence and structure of a large body of proteins are becoming increasingly available. It is desirable to explore mathematical tools for ecient extraction of information from such sources. The principles of graph theory, which was earlier applied in elds such as electrical engineering and computer networks are now being adopted to investigate protein structure, folding, stability, function and dynamics. This review deals with a brief account of relevant graphs and graph theoretic concepts. The concepts of protein graph construction are discussed. The manner in which graphs are analyzed and parameters relevant to protein structure are extracted, are explained. The structural and biological information derived from protein structures using these methods is presented.

The Chaperones Hsp90 and Cdc37 Mediate the Maturation and Stabilization of Protein Kinase C through a Conserved PXXP Motif in the C-terminal Tail

The life cycle of protein kinase C (PKC) is tightly controlled by mechanisms that mature the enzyme, sustain the activation-competent enzyme, and degrade the enzyme. Here we show that a conserved PXXP motif (Kannan, N., Haste, N., Taylor, S. S., and Neuwald, A. F. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 1272–1277), in the C-terminal tail of AGC (c-AMP-dependent protein kinase/protein kinase G/protein kinase C) kinases, controls the processing phosphorylation of conventional and novel PKC isozymes, a required step in the maturation of the enzyme into a signaling-competent species. Mutation of both Pro-616 and Pro-619 to Ala in the conventional PKC βII abolishes the phosphorylation and activity of the kinase. Co-immunoprecipitation studies reveal that conventional and novel, but not atypical, PKC isozymes bind the chaperones Hsp90 and Cdc37 through a PXXP-dependent mechanism. Inhibitors of Hsp90 and Cdc37 significantly reduce the rate of processing phosphorylation of PKC. Of the two C-terminal sites processed by phosphorylation, the hydrophobic motif, but not the turn motif, is regulated by Hsp90. Overlay of purified Hsp90 onto a peptide array containing peptides covering the catalytic domain of PKC βII identified regions surrounding the PXXP segment, but not the PXXP motif itself, as major binding determinants for Hsp90. These Hsp90-binding regions, however, are tethered to the C-terminal tail via a “molecular clamp” formed between the PXXP motif and a conserved Tyr (Tyr-446) in the αE-helix. Disruption of the clamp by mutation of the Tyr to Ala recapitulates the phosphorylation defect of mutating the PXXP motif. These data are consistent with a model in which a molecular clamp created by the PXXP motif in the C-terminal tail and determinants in the αE-helix of the catalytic domain allows the chaperones Hsp90 and Cdc37 to bind newly synthesized PKC, a required event in the processing of PKC by phosphorylation.

Deciphering the Structural Basis of Eukaryotic Protein Kinase Regulation

Biochemical and structural analysis of two features of kinase structure, the “R-spine” and “Shell,” afford a detailed insight into the regulation of eukaryotic protein kinases.

Evolutionary constraints associated with functional specificity of the CMGC protein kinases MAPK, CDK, GSK, SRPK, DYRK, and CK2α

Amino acid residues associated with functional specificity of cyclin‐dependent kinases (CDKs), mitogen‐activated protein kinases (MAPKs), glycogen synthase kinases (GSKs), and CDK‐like kinases (CLKs), which are collectively termed the CMGC group, were identified by categorizing and quantifying the selective constraints acting upon these proteins during evolution. Many constraints specific to CMGC kinases correspond to residues between the N‐terminal end of the activation segment and a CMGC‐conserved insert segment associated with coprotein binding. The strongest such constraint is imposed on a “CMGC‐arginine” near the substrate phosphorylation site with a side chain that plays a role both in substrate recognition and in kinase activation. Two nearby buried waters, which are also present in non‐CMGC kinases, typically position the main chain of this arginine relative to the catalytic loop. These and other CMGC‐specific features suggest a structural linkage between coprotein binding, substrate recognition, and kinase activation. Constraints specific to individual subfamilies point to mechanisms for CMGC kinase specialization. Within casein kinase 2α (CK2α), for example, the binding of one of the buried waters appears prohibited by the side chain of a leucine that is highly conserved within CK2α and that, along with substitution of lysine for the CMGC‐arginine, may contribute to the broad substrate specificity of CK2α by relaxing characteristically conserved, precise interactions near the active site. This leucine is replaced by a conserved isoleucine or valine in other CMGC kinases, thereby illustrating the potential functional significance of subtle amino acid substitutions. Analysis of other CMGC kinases similarly suggests candidate family‐specific residues for experimental follow‐up.

Evolution of allostery in the cyclic nucleotide binding module

BackgroundThe cyclic nucleotide binding (CNB) domain regulates signaling pathways in both eukaryotes and prokaryotes. In this study, we analyze the evolutionary information embedded in genomic sequences to explore the diversity of signaling through the CNB domain and also how the CNB domain elicits a cellular response upon binding to cAMP.ResultsIdentification and classification of CNB domains in Global Ocean Sampling and other protein sequences reveals that they typically are fused to a wide variety of functional domains. CNB domains have undergone major sequence variation during evolution. In particular, the sequence motif that anchors the cAMP phosphate (termed the PBC motif) is strikingly different in some families. This variation may contribute to ligand specificity inasmuch as members of the prokaryotic cooA family, for example, harbor a CNB domain that contains a non-canonical PBC motif and that binds a heme ligand in the cAMP binding pocket. Statistical comparison of the functional constraints imposed on the canonical and non-canonical PBC containing sequences reveals that a key arginine, which coordinates with the cAMP phosphate, has co-evolved with a glycine in a distal β2-β3 loop that allosterically couples cAMP binding to distal regulatory sites.ConclusionOur analysis suggests that CNB domains have evolved as a scaffold to sense a wide variety of second messenger signals. Based on sequence, structural and biochemical data, we propose a mechanism for allosteric regulation by CNB domains.

Global Analysis of Protein Expression and Phosphorylation of Three Stages of Plasmodium falciparum Intraerythrocytic Development

During asexual intraerythrocytic development, Plasmodium falciparum diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the Plasmodium asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the Plasmodium proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase-substrate pairs operative in various signaling networks in the parasite.

Congenital disease SNPs target lineage specific structural elements in protein kinases

The catalytic domain of protein kinases harbors a large number of disease-causing single nucleotide polymorphisms (SNPs) and common or neutral SNPs that are not known or hypothesized to be associated with any disease. Distinguishing these two types of polymorphisms is critical in accurately predicting the causative role of SNPs in both candidate gene and genome-wide association studies. In this study, we have analyzed the structural location of common and disease-associated SNPs in the catalytic domain of protein kinases and find that, although common SNPs are randomly distributed within the catalytic core, known disease SNPs consistently map to regulatory and substrate binding regions. In particular, a buried side-chain network that anchors the flexible activation loop to the catalytic core is frequently mutated in disease patients. This network was recently shown to be absent in distantly related eukaryotic-like kinases, which lack an exaggerated activation loop and, presumably, are not regulated by phosphorylation.

Structural and evolutionary adaptation of rhoptry kinases and pseudokinases, a family of coccidian virulence factors

BackgroundThe widespread protozoan parasite Toxoplasma gondii interferes with host cell functions by exporting the contents of a unique apical organelle, the rhoptry. Among the mix of secreted proteins are an expanded, lineage-specific family of protein kinases termed rhoptry kinases (ROPKs), several of which have been shown to be key virulence factors, including the pseudokinase ROP5. The extent and details of the diversification of this protein family are poorly understood.ResultsIn this study, we comprehensively catalogued the ROPK family in the genomes of Toxoplasma gondii, Neospora caninum and Eimeria tenella, as well as portions of the unfinished genome of Sarcocystis neurona, and classified the identified genes into 42 distinct subfamilies. We systematically compared the rhoptry kinase protein sequences and structures to each other and to the broader superfamily of eukaryotic protein kinases to study the patterns of diversification and neofunctionalization in the ROPK family and its subfamilies. We identified three ROPK sub-clades of particular interest: those bearing a structurally conserved N-terminal extension to the kinase domain (NTE), an E. tenella-specific expansion, and a basal cluster including ROP35 and BPK1 that we term ROPKL. Structural analysis in light of the solved structures ROP2, ROP5, ROP8 and in comparison to typical eukaryotic protein kinases revealed ROPK-specific conservation patterns in two key regions of the kinase domain, surrounding a ROPK-conserved insert in the kinase hinge region and a disulfide bridge in the kinase substrate-binding lobe. We also examined conservation patterns specific to the NTE-bearing clade. We discuss the possible functional consequences of each.ConclusionsOur work sheds light on several important but previously unrecognized features shared among rhoptry kinases, as well as the essential differences between active and degenerate protein kinases. We identify the most distinctive ROPK-specific features conserved across both active kinases and pseudokinases, and discuss these in terms of sequence motifs, evolutionary context, structural impact and potential functional relevance.By characterizing the proteins that enable these parasites to invade the host cell and co-opt its signaling mechanisms, we provide guidance on potential therapeutic targets for the diseases caused by coccidian parasites.