Understanding the Mechanism of Direct Activation of AMP-Kinase: Toward a Fine Allosteric Tuning of the Kinase Activity.

Mammalian AMP-activated protein kinase (AMPK) is a Ser/Thr protein kinase with a key role as a sensor in cellular energy homeostasis. It has a major role in numerous metabolic disorders, such as type 2 diabetes, obesity, and cancer, and hence it has gained progressive interest as a potential therapeutic target. AMPK is a heterotrimeric enzyme composed by an α-catalytic subunit and two regulatory subunits, ß and γ. It is regulated by several mechanisms, including indirect activators such as metformin and direct activators such as compound A-769662. The crystal structure of AMPK bound to A-769662 has been recently reported, suggesting a hypothetical allosteric mechanism of AMPK activation assisted by phosphorylated Ser108 at the ß-subunit. Here, we have studied the direct activation mechanism of A-769662 by means of molecular dynamics simulations, suggesting that the activator may act as a glue, coupling the dynamical motion of the ß-subunit and the N-terminal domain of the α-subunit, and assisting the preorganization of the ATP-binding site. This is achieved through the formation of an allosteric network that connects the activator and ATP-binding sites, particularly through key interactions formed between αAsp88 and ßArg83 and between ßpSer108 and αLys29. Overall, these studies shed light into key mechanistic determinants of the allosteric regulation of this cellular energy sensor, and pave the way for the fine-tuning of the rational design of direct activators of this cellular energy sensor.

Structural and functional features of enzymes of Mycobacterium tuberculosis peptidoglycan biosynthesis as targets for drug development.

Tuberculosis (TB) is the second leading cause of human mortality from infectious diseases worldwide. The WHO reported 1.3 million deaths and 8.6 million new cases of TB in 2012. Mycobacterium tuberculosis (M. tuberculosis), the infectious bacteria that causes TB, is encapsulated by a thick and robust cell wall. The innermost segment of the cell wall is comprised of peptidoglycan, a layer that is required for survival and growth of the pathogen. Enzymes that catalyse biosynthesis of the peptidoglycan are essential and are therefore attractive targets for discovery of novel antibiotics as humans lack similar enzymes making it possible to selectively target bacteria only. In this paper, we have reviewed the structures and functions of enzymes GlmS, GlmM, GlmU, MurA, MurB, MurC, MurD, MurE and MurF from M. tuberculosis that are involved in peptidoglycan biosynthesis. In addition, we report homology modelled 3D structures of those key enzymes from M. tuberculosis of which the structures are still unknown. We demonstrated that natural substrates can be successfully docked into the active sites of the GlmS and GlmU respectively. It is therefore expected that the models and the data provided herein will facilitate translational research to develop new drugs to treat TB.

Homology modeling, molecular dynamics and QM/MM study of the regulatory protein PhoP from Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis is a facultatively intracellular Gram-positive bacterium that causes caseous lymphadenitis, principally in sheep and goats, though sometimes in other species of animals, leading to considerable economic losses. This pathogen has a TCS known as PhoPR, which consists of a sensory histidine kinase protein (PhoR) and an intracellular response regulator protein (PhoP). This system is involved in the regulation of proteins present in various processes, including virulence. The regulation is activated by PhoP protein phosphorylation, an event that requires a magnesium (Mg(2+)) ion. Here we describe the 3D structure of the regulatory response protein (PhoP) of C. pseudotuberculosis through molecular modeling by homology. The model generated provides the first structural information on a full-length member of the OmpR/PhoP subfamily. Classical molecular dynamics was used to investigate the stability of the model. In addition, we used quantum mechanical/molecular mechanical techniques to perform (internal, potential) energy optimizations to determine the interaction energy between the Mg(2+) ion and the structure of the PhoP protein. Analysis of the interaction energy residue by residue shows that Asp-16 and Asp-59 play an important role in the protein-Mg(2+) ion interactions. These results may be useful for the future development of a new vaccine against tuberculosis based on genetic attenuation via a point mutation that results in the polar residue Asp-16 and/or Asp-59 being replaced with a nonpolar residue in the DNA-binding domain of PhoP of C. pseudotuberculosis.