Structure of the Mg-Chelatase Cofactor GUN4 Reveals a Novel Hand-Shaped Fold for Porphyrin Binding

Abstract

In plants, the accumulation of the chlorophyll precursor Mg-protoporphyrin IX (Mg-Proto) in the plastid regulates the expression of a number of nuclear genes with functions related to photosynthesis. Analysis of the plastid-to-nucleus signaling activity of Mg-Proto in Arabidopsis thaliana led to the discovery of GUN4, a novel porphyrin-binding protein that also dramatically enhances the activity of Mg-chelatase, the enzyme that synthesizes Mg-Proto. GUN4 may also play a role in both photoprotection and the cellular shuttling of tetrapyrroles. Here we report a 1.78-Å resolution crystal structure of Synechocystis GUN4, in which the porphyrin-binding domain adopts a unique three dimensional fold with a “cupped hand” shape. Biophysical and biochemical analyses revealed the specific site of interaction between GUN4 and Mg-Proto and the energetic determinants for the GUN4 • Mg-Proto interaction. Our data support a novel protective function for GUN4 in tetrapyrrole trafficking. The combined structural and energetic analyses presented herein form the physical-chemical basis for understanding GUN4 biological activity, including its role in the stimulation of Mg-chelatase activity, as well as in Mg-Proto retrograde signaling.

Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway

“…1 and Ref. 13). However, it should be noted that docking of MgP into the binding pocket as proposed in Ref.…”

“…Synechocystis and T. elongatus WT structures, however, show large differences in orientation of ␣2/␣3 and ␣6/␣7 loops ( Fig. 1), part of the highly conserved Gun4 core domain (13). Based on an extensive analysis of site-directed Synechocystis Gun4 mutant proteins and NMR chemical shift measurements, this core domain was proposed as the porphyrin binding pocket ( Fig.…”

“…However, it should be noted that docking of MgP into the binding pocket as proposed in Ref. 13 is not possible for Synechocystis Gun4. The tight packing derived from the above loop orientations introduces severe steric clashes that return no bound poses from standard docking approaches.…”

“…In bacteriochlorophyll-producing bacteria, these three subunits are sufficient for a highly active MgCh enzyme when assayed in vitro (10). In contrast, MgCh from cyanobacteria, algae, and plants requires a protein called Gun4 that strongly enhances MgCh activity in vitro (11)(12)(13) and very likely also in vivo (14,15).…”

“…Gun4 was shown to bind various porphyrins, exhibiting the highest affinity to MgP, the product of MgCh enzyme, and a weaker affinity to P IX (13,16). There is evidence that Gun4 physically interacts with ChlH (11,14,17), which is the porphyrin-binding MgCh subunit that presumably also contains the active site for chelation (18).…”