Ferredoxin:NADPH oxidoreductase is recruited to thylakoids by binding to a polyproline type II helix in a pH-dependent manner

Abstract

Ferredoxin:NADPH oxidoreductase (FNR) is a key enzyme of photosynthetic electron transport required for generation of reduction equivalents. Recently, two proteins were found to be involved in membrane-anchoring of FNR by specific interaction via a conserved Ser/Pro-rich motif: Tic62 and Trol. Our crystallographic study reveals that the FNR-binding motif, which forms a polyproline type II helix, induces self-assembly of two FNR monomers into a backto-back dimer. Because binding occurs opposite to the FNR active sites, its activity is not affected by the interaction. Surface plasmon resonance analyses disclose a high affinity of FNR to the binding motif, which is strongly increased under acidic conditions. The pH of the chloroplast stroma changes dependent on the light conditions from neutral to slightly acidic in complete darkness or to alkaline at saturating light conditions. Recruiting of FNR to the thylakoids could therefore represent a regulatory mechanism to adapt FNR availability/activity to photosynthetic electron flow.polyproline helix | proline recognition domain | protein-protein interaction | Tic62

N-Terminal Structure of Maize Ferredoxin:NADP+ Reductase Determines Recruitment into Different Thylakoid Membrane Complexes

“…Membranes from Zm-FNR3 lines were not noticeably enriched, and relative staining of TROL and Tic62 complexes was similar to that seen in untransformed Arabidopsis. The kinetics of FNR binding to a motif conserved between TROL and Tic62 are well described (Alte et al, 2010), and the data in Figure 2D now indicate there may be differences between the mechanism of FNR interaction with TROL and Tic62.…”

“…The orientation of Zm-FNR1 and Zm-FNR2 subunits in the homodimer is very similar to that in the cocrystal of pea (Pisum sativum) FNR with a 27-amino acid fragment of Tic62 (Alte et al, 2010). By contrast, the structure of the Zm-FNR3 homodimer is much more open, with the potential Tic62 binding surfaces further apart.…”

“…This is consistent with the poor membrane association behavior of Zm-FNR3. Interestingly, in the Zm-FNR1 homodimer, the N termini of two adjacent Zm-FNR1 molecules are inserted at the interface of the homodimer ( Figure 3C), occupying the space reported to be filled by the Tic62 peptide ( Figure 3E; coordinates taken from Alte et al, 2010). In addition to N-terminal differences, there is variation between the charge distribution at the homodimer-forming surface of FNR proteins ( Figures 3C and 3D).…”

“…TROL mutants are impaired in photosynthesis (Juric et al, 2009), and Tic62 has no direct photosynthetic role but seems rather to stabilize FNR (Benz et al, 2009). An FNR binding domain containing a Ser/Pro-rich motif has been identified in both TROL and Tic62, and its interaction with FNR has recently been thoroughly characterized (Alte et al, 2010). In the crystal structure, FNR dimerizes around this Ser/Pro-rich motif.…”

“…These roles in different electron transport pathways have led to intense research interest in the mechanism and location of FNR binding to the thylakoid membrane (Andersen et al, 1992;Jose Quiles and Cuello, 1998;Zhang et al, 2001;Peng et al, 2008;Benz et al, 2009;Juric et al, 2009;Alte et al, 2010;Baniulis et al, 2011). Two thylakoid FNR binding proteins named TROL and Tic62 have been identified in Arabidopsis thaliana.…”