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

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

Quantitative Analysis of the GUN4 Stimulation of Mg²⁺ Incorporation into Deutero

<div><p>(A) The <i>K</i>_m values for GUN4 assisted Mg-Deutero biosynthesis were determined using both 0.2 μm (red) and 0.4 μM SynGUN4 (green). The resultant values contrast with those obtained for Mg²⁺ incorporation by the Mg-chelatase complex in the absence of SynGUN4 (blue).</p> <p>(B) Relative <i>K</i>_m values (compared to wild-type SynGUN4) were determined for each SynGun4 mutant previously examined for Deutero binding.</p> <p>(C) Rendered ribbon diagrams of orthogonal views of the SynGUN4 core domain with the relative <i>K</i>_m values of each SynGUN4 mutant mapped onto the structure. The color-coded scale for each mutation's effect on Mg²⁺ incorporation is shown at the bottom. Several mutants that altered Mg²⁺ incorporation activity but previously did not affect binding to deuteroporphyrin IX were uncovered including a R217A mutation and the Trp129 and Tyr131 residing on the α2/α3 loop. Mutants of Val218, the later of which is critical for binding Deutero but not for binding Mg-Deutero showed no effect on chelatase activity while mutants of Ala219, the later of which is essential for binding to Mg-Deutero, completely failed to stimulate Mg-chelatase activity. Only those mutant SynGUN4s exhibiting a greater than 10-fold change in <i>K</i>_m are labeled. Shown in black are residues that, when mutated to alanine, failed to produce properly folded protein upon expression in E. coli.</p></div

Overall Structure of <i>Synechocystis</i> GUN4

<div><p>(A) Orthogonal views of the crystal structure of the full length (residues 1–233) <i>Synechocystis</i> GUN4 protein (SynGUN4). Helices are shown as red cylinders and loop regions are displayed as gray loops. SynGUN4 contains two distinct domains linked by a flexible loop. The helices of the N-terminal domain are labeled with apostrophes to distinguish them from the helices making up the C-terminal domain. All structure figures were made with MOLSCRIPT [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030151#pbio-0030151-b57" target="_blank">57</a>] and POV-Ray (<a href="http://www.povray.org" target="_blank">http://www.povray.org</a>).</p> <p>(B) Orthogonal views of the GRASP [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030151#pbio-0030151-b58" target="_blank">58</a>] representation of the SynGUN4 solvent-accessible surface colored to approximately reflect the underlying electrostatic potential, where blue is positive, red is negative, and white is neutral.</p></div

Sequence Alignment of GUN4 and GUN4-like Proteins

<div><p>(A) Alignment of the N-terminal portions of GUN4 family members whose N-terminal domains show sequence homology to SynGUN4. Residues contributing to the hydrophobic core of the five-helix bundle are highlighted (pink). GUN4 sequences isolated from plants thus far all have a plastid transit peptide in place of the N-terminal domain found in SynGUN4. The Chlamydomonas reinhardtii sequence was derived from sequence data produced by the United States Department of Energy Joint Genome Institute (<a href="http://www.jgi.doe.gov/" target="_blank">http://www.jgi.doe.gov/</a>). The N-terminal sequence of C. reinhardtii is not yet known but it most likely contains a chloroplast transit peptide.</p> <p>(B) Sequence alignment of possible GUN4 core domains. Residues that form the “palm” of the “cupped hand” are highlighted in pink. Residues from the α6/α7 loop that structure the loop and protrude into the core are highlighted in yellow. Arg214 and Arg217, predicted to be important for binding to porphyrins, are highlighted in blue. Residues that disrupt proper folding when mutated and expressed in E. coli are denoted by an asterisk (*).</p></div

Analysis of SynGUN4 by NMR

<div><p>(A) Comparison of spectra obtained from ¹H-¹⁵N TROSY experiments of SynGUN4 in the absence (black) and presence (red) of 2 mM deuteroporphyrin.</p> <p>(B) Normalized chemical shifts for those ¹H-¹⁵N cross peaks whose positions change in the presence of 2 mM deuteroporphyrin. In general, the largest shifts cluster for residues on the α6/α7 loop. The remaining positions with significant chemical shifts reside on the “greasy palm” region of SynGUN4.</p> <p>(C) Rendered ribbon diagram of the Gun4 core domain with the position of the shifting ¹H-¹⁵N cross peaks mapped onto the backbone structure of SynGUN4. The magnitude of the chemical shift changes shown corresponds to the color bar at the bottom. Briefly, shifts larger than 2.5 parts per million (ppm) are shown in red, shifts between 2 and 2.5 ppm are shown in orange, shifts between 1.5 and 2 ppm are shown in yellow, and shifts of 1.5 ppm and less are shown in green.</p></div

Model of a Putative SynGUN4 Porphyrin Complex Compared to an Experimentally Determined Structure for Ferrochelatase Bound to NMMP

<div><p>(A) Comparison of the crystal structure of the B. subtilis ferrochelatase bound to NMMP to the model of the SynGUN4 core domain bound to Mg-Proto. The SynGUN4 core domain • Mg-Proto model was generated by GOLD [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030151#pbio-0030151-b54" target="_blank">54</a>]. The carboxylic acid moieties of the porphyrin were staggered between the δ-guanido side chains of Arg214 and Arg217. The position of the arginine loop used to tether the carboxyl moieties of the porphyrin bound to ferrochelatase served as the fixed point for the structural alignment of SynGUN4 and ferrochelatase.</p> <p>(B) Close-up view of the structural alignment between Mg-Proto (gold) and NMMP (lavender). Attempts to strictly superimpose all of the atoms of the two porphyrins resulted in at least one corner of the porphyrin scaffold residing out of the plane defined by the flat Mg-Proto complex, because of the pucker of NMMP.</p></div

Quantitative Analysis of Porphyrin Binding by SynGUN4

<div><p>(A) Comparison of the binding of SynGUN4 to analogs of both Proto and Mg-Proto. Both Mg-Deutero and Deutero quench endogenous tryptophan fluorescence upon binding (inset). A single binding site was assumed for the fitted line.</p> <p>(B) Relative dissociation constants were determined for each mutant and compared to the wild-type dissociation constant for both Deutero (red bars) and Mg-Deutero (green bars). The difference between these two sets of constants was calculated (blue bars).</p> <p>(C) Rendered ribbon diagram of the GUN4 core domain with the relative dissociation constants of each mutant for Deutero mapped onto the structure. While in some cases several different amino acid replacements were tested at particular positions, only the results obtained for the alanine mutations are mapped on the backbone structure shown. The x-fold change in the magnitude of the affinity of Deutero for each mutant is color-coded, as depicted by the scale shown at the bottom. Most amino acid changes did not alter binding affinity, as shown by the preponderance of light blue. Of the mutations that measurably alter binding affinity, the majority reside on the “greasy palm” of the GUN4 core domain. Several other energetic hotspots reside on the α2/α3 and α6/α7 loops. Positions of mutations that exhibit a greater than 10-fold decrease in affinity are labeled. Positions colored black failed to produce properly folded protein when mutated to alanine and expressed in E. coli.</p> <p>(D) Rendered ribbon diagram of the GUN4 core domain with the relative dissociation constants of each mutant for Mg-Deutero mapped onto the structure. Color coding is the same as for (C). In contrast to Deutero binding, many more mutants alter in vitro binding as shown by the lesser amount of light blue and the prominence of green and yellow color coding.</p></div

Close-Up View of the GUN4 Core Domain's “Cupped Hand” Architecture

<div><p>(A) Rendered skeletal view of the GUN4 core domain. Helices are shown as red cylinders, and coiled regions are depicted as gray loops. The overall shape resembles that of a “cupped hand.”</p> <p>(B) Rendered view of the solvent-accessible surface of the GUN4 core domain, colored gold. The α6/α7 loop is colored gray and is bound by the remainder of the domain. The “cupped hand” grips this loop.</p></div