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The Fic domain was recently shown to catalyze AMPylation--the transfer of AMP from ATP to hydroxyl side chains of diverse eukaryotic proteins, ranging from RhoGTPases to chaperon BiP. We have carried out a series of explicit solvent molecular dynamics (MD) simulations up to 1 μs duration on the apo, holo, and substrate/product bound IbpA Fic domain (IbpAFic2). Simulations on holo-IbpAFic2 revealed that binding of Mg2+ to α and β phosphates is crucial for preserving catalytically important contacts involving ATP. Comparative analysis of the MD trajectories demonstrated how binding of ATP allosterically induces conformational changes in the distal switch II binding region of Fic domains thereby aiding in substrate recognition. Our simulations have also identified crucial aromatic-aromatic interactions which stabilize the orientation of the catalytic histidine for inline nucleophilic attack during AMPylation, thus providing a structural basis for the evolutionary conservation of these aromatic residue pairs in Fic domains. On the basis of analysis of interacting interface residue pairs that persist over the microsecond trajectory, we identified a tetrapeptide stretch involved in substrate recognition. The structure-based genome-wide search revealed a distinct conservation pattern for this segment in different Fic subfamilies, further supporting its proposed role in substrate recognition. In addition, combined use of simulations and phylogenetic analysis has helped in the discovery of a new subfamily of Fic proteins that harbor a conserved Lys/Arg in place of the inhibitory Glu of the regulatory helix. We propose the novel possibility of auto-enhancement of AMPylation activity in this new subfamily via the movement of regulatory helix, in contrast to auto-inhibition seen in most Fic proteins. [ABSTRACT FROM AUTHOR]

Mycobacterial Ser/Thr kinase, PknB, is essential for the growth of the pathogen. Unphosphorylated PknB is catalytically inactive, and its activation requires autophosphorylation of Thr residues on the activation loop. Autophosphorylation can in principle take place via two distinct mechanisms. Intermolecular trans autophosphorylation involves dimerization and phosphorylation of the activation loop of one chain in the catalytic pocket of the other chain. On the other hand, intramolecular cis autophosphorylation involves phosphorylation of the activation loop of the kinases in its own catalytic pocket within a monomer. On the basis of the crystal structure of PknB in the front-to-front dimeric form, it is currently believed that activation of PknB involves trans autophosphorylation. However, because of the lack of coordinates of the activation loop in the crystal structures, atomic details of the conformational changes associated with activation are yet to be deciphered. Therefore, to understand the conformational transitions associated with activation via autophosphorylation, a series of explicit solvent molecular dynamics simulations with a duration of 1 µs have been performed on each of the phosphorylated and nonphosphorylated forms of the PknB catalytic domain in monomeric and dimeric states. Simulations on phosphorylated PknB revealed a differential network of crucial electrostatic and hydrophobic residues that stabilize the phosphorylated form in the active conformation. Interestingly, in our simulations on nonphosphorylated monomers, the activation loop was observed to fold into its own active site, thereby opening the novel possibility of activation through intramolecular cis autophosphorylation. Thus, our simulations suggest that autophosphorylation of PknB might also involve cis initiation followed by trans amplification as reported for other eukaryotic kinases based on recent reaction kinetics studies. [ABSTRACT FROM AUTHOR]

This article focuses on the precursors of polyketide biosynthesis. The study of bioactive natural products has undergone rapid advancement with the cloning and sequencing of large numbers of gene clusters. Polyketide synthases utilize acyl-coenzyme A (CoA) precursors and synthesize polyketides by repetitive decarboxylative condensations. The potential of these proteins to produce varied CoA precursors and their utilization in polyketide biosynthesis have not been investigated. Acyl-activating enzymes proteins provide an attractive route to expand the precursors available for polyketide biosynthesis.