About
We recently demonstrated the existence of a phosphorylation-dependent control of glutamate metabolism in mycobacteria, in collaboration with H. O’Hare’s group in Leicester (O’Hare et al., 2008). Our results were supported by similar observations in Corynebacterium glutamicum (Niebisch et al., 2006; Bott, 2007) and have been further corroborated by the work from Steve Smerdon’s team at NIMR, London (Nott et al., 2009). Pivot of this metabolic regulation network is the FHA protein GarA (Rv1827 in M. tuberculosis). We have shown that, contrarily to a widely accepted view about FHA proteins, GarA does not merely act as a pThr-recognition module, but also interacts with several metabolic enzymes through phosphoindependent interactions. When unphosphorylated, GarA acts an inhibitor of Kgd (α-ketoglutarate decarboxylase) and GDH (glutamate dehydrogenase), whereas it activates the α-subunit of the glutamate synthase complex (GS; Nott et al., 2009) overall leading to the accumulation of glutamate. Once GarA is phosphorylated (mainly by PknG in vivo, but possibly also by PknB or other Ser/Thr kinases), the self-recognition of the phoshorylated Thr by the FHA domain (England et al., 2008) prevents the interaction of GarA with its downstream partners. We are investigating the basis of regulation of the enzymatic activity of Kgd, the E1o component of the α-ketoglutarate dehydrogenase complex KDH, since it represents a key element of the TCA cycle positioned at a crucial metabolic crossroad between energy production and nitrogen assimilation. Our approach uses an ensemble of structural, biochemical and biophysical techniques in order to elucidate how GarA is able to recognize its ‘targets’ specifically (they are not phosphorylated) and to regulate their activity.
We are also interested in determining the signal that triggers the phosphorylation of GarA and therefore its ‘switching-off’. Although the involvement of PknG (and possibly PknB) in phosphorylating GarA is now well established, several issues still need to be addressed. First, the physiological role of PknG is still controversial. This soluble kinase, with no predicted TM segment, was proposed to be involved into the control of glutamate metabolism (Cowley et al., 2004) and, shortly after, to be secreted into infected macrophages, where it would interfere with PKCα signalling to inhibit phagosome-lysosome fusion and enhance mycobacterial survival inside the host (Walburger et al., 2004; Chaurasiya and Srivastava, 2010). Although no clues about the possible secretion mechanism are available to date, the activating signal, either in the eukaryotic phagosome or inside the mycobacterial cell, remains unknown. None of the mechanisms observed in other M. tuberculosis Ser/Thr kinases and shared with eukaryotic counterparts (autophoshorylation on the activation loop, back-to-back dimerization) appear to operate in PknG. The structure of the kinase (Scherr et al., 2007) shows an homodimer, in which dimerization does not involve the catalytic domain in back-to-back (as observed for PknB and PknE) but the C-terminal TPR-domain (pdb id 2pzi). Moreover, a metal-binding rubredoxin-like domain is present at the N-terminus, and mutation of the four cysteines to serine resulted in total loss of activity (Scherr et al., 2007). A role of this domain in sensing redox conditions can be hypothesized, but experimental evidence is still missing. We are thus carrying out detailed biochemical, biophysical and structural studies of different truncated PknG constructs, to investigate the activation mechanism of this kinase and the possible role of the bound metal in signal sensing.
Funding: NM4TB, ANR (TBKIN project)
Collaborations:Helen O’Hare, Department of Infection, Immunity and Inflammation, University of Leicester (UK)