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

春天的风筝

1 Chemical Biology and Proteomics Laboratory, Salk Institute for Biological Studies, La Jolla, California, United States of America,2 Howard Hughes Medical Institute, Plant Molecular and Cellular Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California, United States of America,3 The Institut de Biologie Structurale, Grenoble, France" X* Q7 M- F$ X4 t6 v% t) g

( d' m& L1 ^; i# N% ]) fIn 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- 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.
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" [: [9 @) W) ?a Current address: Department of Neurobiology and Behavior, State University of New York, Stony Brook, New York, United States of America4 b% i: K) `- T9 c

3 w8 w$ F3 `" _# db Current address: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan, United States of America
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Introduction  ?- R5 `7 o5 R( H" `, G
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Organelle function is controlled primarily by the regulation of nuclear gene expression in response to developmental and environmental cues. In turn, organelles signal to the nucleus, in a process termed retrograde signaling, to coordinate the biological activities of the two subcellular compartments. For example, in animals and yeast, mitochondria-to-nucleus and ER-to-nucleus signaling have a dramatic impact on cellular activities under a variety of conditions [1,2]. In plants, plastid-to-nucleus signaling significantly alters the expression of nuclear genes that encode chloroplast-localized proteins involved in photosynthesis and leaf morphogenesis [3,4,5,6]. Therefore, signals originating from plastids play major roles in photoautotrophic growth.
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: N% N' H. R$ I  LGenetic and physiological studies indicate that the accumulation of the chlorophyll precursors Mg-protoporphyrin IX (Mg-Proto) and Mg-protoporphyrin IX monomethyl ester (Mg-ProtoMe) act as a plastid signal that regulates nuclear gene expression in plants and algae [6,7,8,9,10,11]. The current hypothesis proposes that the plastid exports Mg-Proto and/or Mg-ProtoMe, which then interact with a cytoplasmic signaling pathway that ultimately regulates nuclear gene expression [6,9,11]. This proposed model is not without precedent; heme, a tetrapyrrole that bears a striking resemblance to Mg-Proto, regulates gene expression in animal, yeast, and bacterial cells by binding to transcription factors or to kinases that regulate translation [12,13,14,15,16]. Moreover, the bulk of cellular heme is produced in chloroplasts, which is then transported to other cellular compartments [16,17]. Because of the molecular similarity between heme and Mg-Proto, it is reasonable to assume that the cellular machinery used to export heme from the chloroplast may be similar to the machinery used for Mg-Proto export.. Z/ e3 Y: T2 [: t: i
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In a search for mutants that affect communication between chloroplasts and the nucleus, a number of mutants, called gun mutants, were identified that have defects in plastid-to-nucleus signaling pathways. These plastid-to-nucleus signaling pathways repress the transcription of nuclear genes that encode proteins active in photosynthesis when chloroplast development is blocked [18,19]. A number of GUN genes were found to encode factors that participate in Mg-Proto metabolism. Among these were subunits of Mg-chelatase, the enzyme that synthesizes Mg-Proto from protoporphyrin IX (Proto), and indeed, we have shown that buildup of Mg-Proto is a signal that regulates nuclear gene expression [6,18]. GUN4 participates in the same Mg-Proto signaling pathway that Mg-chelatase does, but GUN4 is not related to any previously described Mg-chelatase subunit or any gene with a known function [20]. Purification of a GUN4 complex from Arabidopsis thylakoids revealed that a fraction of GUN4 is tightly associated with GUN5 [20], also called ChlH, which is the 140-kDa subunit of Mg-chelatase [18,21]. Although GUN4 is not essential for Mg-Proto synthesis in vitro or in Arabidopsis, GUN4 is required for chlorophyll accumulation in Arabidopsis under normal growth conditions, GUN4 binds porphyrins in vitro, and GUN4 stimulates Mg-chelatase in vitro [20].
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Thus, GUN4, like GUN5, is a key participant in the generation of a plastid signal. Also, because GUN4 appears to be monomeric or associated with heterogeneous complexes in fractionated chloroplasts, and because GUN4 binds to Mg-Proto more tightly than GUN5 in Synechocystis, it is reasonable to expect that GUN4 might perform additional functions in porphyrin metabolism [20]. For instance, GUN4 might participate in Mg-Proto trafficking or shield Mg-Proto from collisions with molecular oxygen and light, collisions that could result in the production of reactive oxygen species. Alternatively, GUN4 might protect Mg-Proto from catabolic enzymes found in the plastid [22], or it might participate in other tetrapyrrole biosynthetic reactions localized to plastids [23].4 k, s4 m2 i- w. l: P" X
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To better understand the porphyrin-binding mechanism and Mg-chelatase stimulatory activity of GUN4, we determined the crystal structure of a GUN4 homolog from the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis 6803) and refined the three-dimensional model to 1.78  resolution. The crystal structure reveals a novel fold that bears no resemblance to known porphyrin-binding proteins. We subsequently used nuclear magnetic resonance (NMR) to map the porphyrin-binding site through analysis of chemical shift data. Quantitative analysis of the putative porphyrin binding site on GUN4 using fluorescence quenching and enzymatic assays has allowed us to determine the energetic contribution of key residues for porphyrin binding, as well as for the enhancement of Mg2  incorporation into metal-free porphyrins. Taken together, these data paint a picture of a novel enzymatic cofactor that enhances Mg-Proto biosynthesis and additionally, may play a role in Mg-Proto shuttling and chemical protection within the cell., j" W7 X) I2 H7 h
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Crystallization, Structure Determination, and Refinement
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/ n0 ]( a% X. i8 T3 \The crystallized protein (residues 1–233) comprises the entire Synechocystis GUN4 (SynGUN4) protein. Full-length protein was shown to be competent for binding to both deuteroporphyrin IX (Deutero) and Mg-deuteroporphyrin IX (Mg-Deutero). It was also shown to significantly enhance Mg2  incorporation into Deutero in the presence of the Synechocystis Mg-chelatase, which contains the ChlD, ChlH, and ChlI subunits [21]. The SynGUN4 structure was solved by multiple isomorphous replacement using crystals soaked with methyl mercury (II) chloride (Hg) and potassium tetrachloroplatinate II-containing compounds. The resultant model was built and refined to a crystallographic Rcryst and Rfree of 22.1% and 25.9%, respectively, using all Hg data extending to 1.78  resolution (Table 1).
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Structure of GUN4
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The crystal structure of SynGUN4 reveals a two-domain protein linked by a 12-residue loop (Figure 1A). The C-terminal domain, which we refer to herein as the GUN4 core domain, is the conserved domain among all GUN4 family members. The N-terminal domain of SynGUN4 is composed of the first five helices of the full-length protein. The 汐1∩ to 汐4∩ helices fold into a right-handed, up-and-down helix bundle, and the addition of the 汐5∩ helix gives the domain an elongated cross-section. Overall, the N-terminal helical bundle is reminiscent in appearance to other entirely helical domains such as the TPR domain [24] or the 14–3-3 domain [25]. The entire helical bundle is held together through a hydrophobic core consisting entirely of centrally located and interdigitated leucines, isoleucines, and valines provided by each helix. In contrast to the N-terminal domain's core, the surface is highly charged (Figure 1B). Structurally, the 汐2∩ and 汐3∩ helices are the linchpins of this tertiary architecture as they bridge one end of the bundle to the other.2 [( h1 f- P* g/ C3 Z
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(A) Orthogonal views of the crystal structure of the full length (residues 1–233) Synechocystis 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 [57] and POV-Ray (http://www.povray.org).
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(B) Orthogonal views of the GRASP [58] 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.0 r6 F# x$ Q" {+ P0 z4 f) w

- t+ N1 K: K) s3 Y! F+ FInterestingly, analysis of the known GUN4 sequences from other organisms shows that the N-terminal region is the most highly variable sequence and is unique to only some of the prokaryotic family members. SynGUN4's N-terminal region shares homology with GUN4 family members from Gracilaria tenuistipitata var. liui, Porphyra purpurea, Nostoc sp. 7120, Trichodesmium erythraeum, Anabaena variabilis, Thermosynechococcus elongatus, and Cyanidium caldarium (Figure 2A). The Cyanidioschyzon merolae sequence lacks the N-terminal extension altogether. Additionally, within the Arabidopsis and rice GUN4 sequences, the N-terminal domain is replaced with a chloroplast transit peptide, which is removed upon import into the chloroplast [26]. What role this N-terminal helical bundle plays within prokaryotes is unknown, but most likely it does not participate in GUN4 functions conserved between prokaryotic and eukaryotic organisms.8 Y& t/ o- X+ H4 V: _5 _* u3 b- [
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(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 (http://www.jgi.doe.gov/). The N-terminal sequence of C. reinhardtii is not yet known but it most likely contains a chloroplast transit peptide.
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(B) Sequence alignment of possible GUN4 core domains. Residues that form the ※palm§ of the ※cupped hand§ are highlighted in pink. Residues from the 汐6/汐7 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 ().6 [  j  R: `1 Q: z  m2 c4 \

5 ~" z" s/ s  L" d9 x3 mThe N-terminal domain links to the C-terminal domain through a long loop (residues 81–93) that connects the 汐5∩ helix of the N-terminal helical bundle to the 汐1 helix within the GUN4 core domain (Figure 2B). The interaction between the two domains is not extensive and is mediated by van der Waal's interactions between Pro51 and Leu53 from the 汐4∩ helix and Trp151 and Leu152 from the 汐4 helix within the C-terminal domain. Additionally, the carbonyl oxygen of Gly101 on the 汐1 helix is within hydrogen bonding distance of Asn60 and Arg63 located on the 汐4∩ helix. In all, a total of 1,183 2 of surface area is buried between the two domains, which suggests that the particular arrangement displayed in the crystal packing may be one of several possible orientations juxtaposing the two domains in solution." ]- M$ z0 l' R. n( @- H6 |
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The C-terminal domain of SynGUN4, the GUN4 core domain, appears to have no currently identified structural homologs, as indicated by a lack of any structural matches from a search of the DALI server [27]. The GUN4 core domain maintains two distinct architectural regions: a highly structured helical section that forms the majority of the domain, and two loops (汐2/汐3 loop and 汐6/汐7 loop), which constitute one side of the domain (Figure 3A). The helices adopt a concave shell shape resembling a ※cupped hand§ on one side of the domain, with the back of the ※hand§ facing toward the N-terminal helical bundle. Helices 汐1 and 汐2 extend like a thumb and index finger to form one side of the ※cupped hand.§ The 汐3 and 汐4 helices compose the middle finger of the hand, with the 汐3/汐4 loop forming a knuckle. The 汐5 and 汐6 and the 汐7 and 汐8 helices form the remaining fingers, respectively, on the opposite side of the ※cupped hand§ arrangement.
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(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.§
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; X2 K( i* ~. \1 B0 b! {(B) Rendered view of the solvent-accessible surface of the GUN4 core domain, colored gold. The 汐6/汐7 loop is colored gray and is bound by the remainder of the domain. The ※cupped hand§ grips this loop.- `0 ?# \  V# d" q9 z& }5 k) G
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In total, the helical section constitutes 70% of the GUN4 core domain. The buried surface within the concave section of the ※cupped hand§ is highly hydrophobic. Phe105 and Leu116 on the 汐2 helix; Val135 and Phe138 from the 汐3 helix; Leu143, Ile146, and Trp150 from the 汐4 helix; Phe160, Val162, Val166, and Trp167 from the 汐5 helix; Phe174, Leu177, and Trp178 from the 汐6 helix; Val218, Ala219, and Tyr223 from the 汐7 helix; and Trp228 from the 汐8 helix form an extensive hydrophobic surface or ※greasy palm§ of the ※cupped hand.§ Lying loosely across this palm is the 汐6/汐7 loop, which is itself very hydrophobic (Figure 3B). The loop is striking in that it lacks any clear secondary structure, yet it very neatly folds in on itself via van der Waal's interactions between hydrophobic residues. Residues Ile181, Trp183, Trp189, Pro193, Phe196, and Trp198 form a hydrophobic groove within which sits Leu207 and Leu209. The extensive hydrophobicity and intricate arrangement of residues suggest that this loop forms a stable structure that would resist unraveling.
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: I9 a; Z, k4 `: u5 |/ w& i" ^In contrast to the intraloop packing, only two residues (Pro208 and Leu210) protrude from the 汐6/汐7 loop into the ※greasy palm§ of the GUN4 core domain's ※cupped hand.§ Mutation of either position to alanine leads to the expression of misfolded protein as determined by inclusion body production in E. coli during attempted purification of recombinant samples (unpublished data). The necessity of both Pro208 and Leu210 in maintaining protein stability is not surprising, given the lack of interaction between the 汐6/汐7 loop and the ※cupped hand.§ Analysis of this region of the structure reveals that the juncture of the 汐6/汐7 loop with the ※greasy palm§ forms an extended hydrophobic surface that is shielded by the ※thumb§ (汐2 helix) and the ※middle finger§ (汐3 and 汐4 helices). In total, this structural design forms a cavity that is hydrophobic in nature with a volume of about 5,000 3. Additionally, analysis of 2Fo-Fc electron density maps contoured at 1考 reveals several well-ordered water molecules within the confines of the greasy palm, which, given the high degree of hydrophobicity of this space, are unusual.
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Mapping the Porphyrin Binding Site5 t. R; U. x( f3 m+ Z

' ]  g( v9 m+ @) H' XIn an effort to determine the binding site for porphyrin within SynGUN4, we used NMR to analyze the full length protein in the absence and presence of Deutero. Comparison of spectra obtained from 1H-15N transverse relaxation-optimized spectroscopy (TROSY) experiments of SynGUN4 in the absence and presence of 1–2 mM Deutero reveals several shifting peaks that were picked with the program CARA (Figure 4A) [28]. Chemical shifts were calculated for those peaks whose position changes in the presence of Deutero. Partial sequence-specific assignment for the backbone atoms of shifting peaks was obtained using TROSY-type triple resonance, and 15N-resolved [1H,1H] nuclear overhauser effect spectroscopy (NOESY) experiments with a 2H, 13C, 15N-labeled sample of SynGUN4 (1–233) (Figure 4B)., X' E* b8 v1 k( G3 X" a( Y) L1 ]! z
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(A) Comparison of spectra obtained from 1H-15N TROSY experiments of SynGUN4 in the absence (black) and presence (red) of 2 mM deuteroporphyrin.- H) D5 y4 D; S3 h  M; n2 u, O, d

" {& M/ P* T6 V: `- c. r! O, |(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 汐6/汐7 loop. The remaining positions with significant chemical shifts reside on the ※greasy palm§ region of SynGUN4.+ Y: ^1 D9 B" t9 ^+ z' \
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(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.
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The vast majority of the residues whose environments drastically change are within the GUN4 core domain (Figure 4C). Of the 13 residues that were found to exhibit significant chemical shift perturbations in the presence of Deutero, only two (Tyr59 and Thr72) are located within the nonconserved N-terminal helical bundle domain. Of the remaining residues, three residues (Val135, Val218, and Trp228) form part of the ※greasy palm,§ six residues (Asn187, Arg191, Trp198, Asp199, Ser201, and Pro208) are localized within the 汐6/汐7 loop, and the remaining two residues (Lys180 and Arg217) form the hinge regions at the ends of the 汐6/汐7 loop. The chemical shifts offer a very precise measure of the chemical environment around the backbone nitrogen atoms at these positions. The observed chemical shift changes upon addition of Deutero suggest that the 汐6/汐7 loop is undergoing a significant conformational change and/or that an electron-rich molecule, such as Deutero, lies within close proximity. As mentioned earlier, the interaction between this 汐6/汐7 loop and the hydrophobic ※greasy palm§ of the GUN4 core domain is not extensive, and rearrangements of either or both necessary to accommodate the porphyrin molecule are feasible in both a dynamic and architectural sense. Taken together, the NMR data and the crystal structure strongly suggest that the porphyrin binding region lies within the pocket formed at the intersection of the 汐6/汐7 loop and the ※greasy palm§ of the GUN4 core domain's ※cupped hand§ region." L- N" V  j' a" V  b5 F' x

  p# w' p8 y4 I3 F* E7 eDeuteroporphyrin IX and Mg-Deuteroporphyrin IX Binding  b! D) W0 p: K- t0 Q4 Q
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First, the affinity of wild-type SynGUN4 for various porphyrins was investigated (Figure 5A). The dissociation constants (Kd) for protoporphyrin analogs, Mg-Deutero (0.449 ± 0.045 μM) and Deutero (0.865 ± 0.146 μM) were measured. Additionally, SynGUN4's dissociation constants for deuteroporphyrin IX 2,4-(4,2) hydroxyethyl-vinyl-(deutero-divinyl) (3.94 ± 0.739 μM), hemin (4.73 ± 1.16 μM), N-methyl mesoporphyrin IX (NMMP) (11.0 ± 0.673 μM) and cobalt (III) protoporphyrin IX (Co-Proto) (2.67 ± 0.856 μM) were determined. SynGUN4 displayed the highest affinity for Mg-Deutero and Deutero with a preference for the metal-bound porphyrin. The reported dissociation constants for the Synechocystis Mg-chelatase enzyme, ChlH, are 1.22 ± 0.420 μM for Deutero and 2.43 ± 0.460 μM for Mg-Deutero [29].1 H! _5 O) l+ v% A5 ?

8 Q1 l5 Z7 ]; |/ o* f(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.; [0 {* u: ?. H

$ S) c! u; a7 ~! r8 L(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).
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% j4 Z# v9 B& p% V) i- c(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 汐2/汐3 and 汐6/汐7 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.# k! e$ G4 e2 b
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(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.
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( x3 e' ^+ h9 v4 u. _Analysis of SynGUN4's affinity for other porphyrins provides a larger context from which to deduce the determinants of binding specificity. Both deuteroporphyrins are smaller than the other porphyrins examined in that they lack two ethylene groups. Hemin and Co-Proto, which closely mimic the size of the naturally occurring substrate Mg-Proto, bind with slightly weaker affinities. This suggests that Mg-Proto and Proto may also bind with slightly weaker affinity then the their Deutero versions. Nonetheless, both hemin and Co-Proto bind as well as deutero-divinyl, which accurately mimics the size of the naturally occurring metal free Proto. This further supports that the less flexible metal bound porphyrins are indeed high affinity ligands for SynGUN4. Interestingly, NMMP, which mimics distorted porphyrins binds with very weak affinity. NMMP is a potent inhibitor of enzymes known to catalyze insertion of metals into porphyrins, including ferrochelatase [21,30]. SynGUN4, however, appears to favor the more planar metal-bound porphyrins.% n) A! N5 T: s* L
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To quantify the energetic contributions of specific residues in SynGUN4 involved in porphyrin binding, a series of putative porphyrin-binding site mutants were made and dissociation constants were measured. Most of the side chains were mutated to alanine; however, at some positions, other amino acids were investigated to explore the importance of electrostatics, side chain volume, or hydrophobicity in greater detail. Endogenous tryptophan fluorescence quenching of SynGUN4 by Deutero and Mg-Deutero was used to determine the Kd for all point mutants for which protein could be expressed and purified (Table 2). Relative values of all the dissociation constants for each mutant were determined by comparing the Kd of each mutant to that measured for wild-type SynGUN4 (Figure 5B).
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Comparison of the relative values for binding to both Deutero and Mg-Deutero shows that the majority of positions affecting binding cluster in neighboring regions of the GUN4 core domain. The positions that appear to be most critical for binding to both porphyrins reside in the 汐2/汐3 and 汐6/汐7 loops, as well as on the faces of the 汐2, 汐3, and 汐7 helices that form significant parts of the ※greasy palm§ within the ※cupped hand§ of SynGUN4. Calculation of the difference between the two sets of the relative Kd values shows that certain residues appear to be more important for binding Deutero instead of Mg-Deutero, and vice versa. For example, Val135 and Val218 appear to be essential for Deutero binding but not for Mg-Deutero binding. Alternatively, Ser125, Gln126, Ile146, Phe160, and A219 appear to be essential for specifically involved in binding Mg-Deutero.
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Comparison of Mg-Deutero binding to Deutero binding shows that binding of the metal-bound porphyrin is much more sensitive to changes within the GUN4 core domain (Figure 5C and 5D). This difference most likely reflects the highly specialized architecture involved in binding the more rigid Mg-Deutero.! l9 Y+ f9 g4 B( ^: Q

+ I) e" I6 j) H) Y5 c1 @! eModeling of Porphyrin Binding
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8 p7 T) }, r% P, w2 wArmed with an energy map of relevant positions for porphyrin binding, we generated a model of SynGUN4 bound to Mg-Proto (Figure 6). In this model, the porphyrin molecule sits over Leu210 within the 汐6/汐7 loop, deep in the ※greasy palm§ of SynGUN4. The solvent-exposed section of the 汐6/汐7 and 汐2/汐3 loops bracket the porphyrin, burying it deep within SynGUN4 core domain. Significantly, the carboxyl moieties of the porphyrin insert between Arg214 and Arg217, which would be complementary to the charge of the carboxylic acid groups extending from the porphyrin scaffold. Analysis of the Bacillus subtilis ferrochelatase structure bound to NMMP shows that this porphyrin-binding chelatase uses a pair of conserved arginines to bind the carboxyl moieties extending off the porphyrin scaffold (Figure 6A). The Arg214 and Arg217 positions within the 汐6/汐7 loop of SynGUN4 closely resemble the arginine motif found on ferrochelatase, suggesting that this motif in SynGUN4 may function in an analogous fashion upon porphyrin binding (Figure 6A).
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$ q1 {" p2 `1 ](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 [54]. The carboxylic acid moieties of the porphyrin were staggered between the 汛-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.& x$ g/ `9 D7 }5 J. k2 v% H

8 A. Y4 r  T7 e* G! ?8 F; [! H) @(B)(Mark A. Verdeciaa, Robert)

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yukun

干细胞从业人员