Adaptation of Aminoacyl-tRNA Synthetase Catalytic Core to Carrier Protein Aminoacylation

Structure

Article Adaptation of Aminoacyl-tRNA Synthetase Catalytic Core to Carrier Protein Aminoacylation Marko Mocibob,1,3 Nives Ivic,2,3 Marija Luic,2 and Ivana Weygand-Durasevic1,* 1Department

of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, 10 000 Zagreb, Croatia ka 54, 10 000 Zagreb, Croatia of Physical Chemistry, Rudjer Boskovic Institute, Bijenic 3These authors contributed equally to this work *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2013.02.017 2Department

SUMMARY

Amino acid:[carrier protein] ligases (aa:CP ligases) are recently discovered enzymes that are highly similar to class II aminoacyl-tRNA synthetases (aaRSs). However, while aaRSs aminoacylate tRNA and supply building blocks for ribosomal translation, aa:CP ligases transfer activated amino acids to the phosphopantetheine group of small carrier proteins. We have solved the crystal structure of an aa:CP ligase complexed with the carrier protein (CP). The CP prosthetic group enters the active site from a different direction than tRNA in class II aaRS complexes through an idiosyncratic tunnel. CP binds to aa:CP ligase in a fundamentally different manner compared to tRNA binding by structurally closely related aaRSs. Based on crystallographic analysis, an enzyme of altered CP specificity was designed, and the mechanism of amino acid transfer to the prosthetic group was proposed. The presented study reveals how a conserved class II aaRS catalytic core can adapt to another function through minor structural alterations.

INTRODUCTION Aminoacyl-tRNA synthetases (aaRSs) are enzymes that ligate specific amino acids to their cognate tRNA molecules (Ibba and So¨ll, 2000). An aaRS first activates the amino acid to form aminoacyl-adenylate and then transfers the amino acid to the 30 -terminal adenosine of the tRNA to be used in ribosomal protein synthesis. Based on two structurally unrelated catalytic core domains, the 20 aaRSs, each corresponding to one of the 20 amino acids, are divided into two classes (Eriani et al., 1990). Recent analyses of aaRS-like proteins lacking the tRNA aminoacylation activities (aaRS paralogs) have revealed their roles in various important biological processes in and beyond translation (Park et al., 2005). We have focused on seryl-tRNA synthetases (SerRSs) because this family of enzymes displays remarkable structural and functional diversity (Bilokapic et al., 2006). The noncanonical functions of SerRS family members are exemplified by their involvement in antibiotic synthesis (Garg et al., 2006) and resistance (Zeng et al., 2009), oxidative-

stress-related mitochondrial processes (Guitart et al., 2010), nonribosomal peptide synthesis (Mocibob et al., 2010) and, most notably, in the regulation of vascular development by vertebrate SerRSs (Kawahara and Stainier, 2009). The latter was first discovered in zebrafish. That discovery was followed by evidence that the C-terminally appended domain of human SerRS, which is dispensable for aminoacylation, directs the synthetase to the nucleus where it attenuates vascular endothelial growth factor A expression (Xu et al., 2012). Crystal structures of SerRS representatives from all domains of life (Fujinaga et al., 1993; Itoh et al., 2008; Rocha et al., 2011) confirm the existence of many evolutionarily conserved features. All SerRSs are dimers, and each subunit consists of an N-terminal tRNA-binding domain and a C-terminal catalytic domain that is also responsible for dimerization. The N-terminal domain is indispensable for tRNA binding, even for the enzyme from mammalian mitochondria, which possesses very unusual serine-specific tRNA acceptors (Chimnaronk et al., 2005). Furthermore, crystallographic, docking, and biochemical experiments have revealed that a tRNA molecule binds across two enzymes’ subunits. The N-terminal domains of the standard (bacterial-type) SerRSs consist of two long antiparallel helices forming a coiled coil (Fujinaga et al., 1993) with characteristic insertions in some SerRSs (Xu et al., 2012; Itoh et al., 2008; Rocha et al., 2011). The exception is an atypical SerRS that functions in methanogenic archaea and does not have the coiled-coil structure. This domain is significantly larger in methanogenictype enzymes. In Methanosarcina barkeri methanogenic-type SerRS (mMbSerRS), the N-terminal domain is composed of a six-stranded antiparallel b sheet capped by a bundle of four short helices (Bilokapic et al., 2006). However, despite the observed structural difference, N-terminal domains of all SerRSs are involved in tRNA binding and are similarly positioned relative to the catalytic domain. Because the crystal structure of methanogenic-type SerRS in complex with tRNA has not been obtained yet, the crucial role of the N-terminal residues in tRNA binding and aminoacylation has been pinpointed by mutagenesis based on a docking model followed by in vivo and in vitro analysis (Jaric et al., 2009). We have also experimentally demonstrated cross-dimer tRNA binding in methanogens (Bilokapic et al., 2009), which seems to be another evolutionary conserved feature in the serine system. Except for mammalian mitochondria, the long variable arm of the tRNASer is recognized by the N-terminal domain of SerRS, while the 30 -end of tRNASer enters the active site of the opposite subunit from the direction of the motif 2 loop. In methanogenic-type

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Structure Carrier Protein Recognition by aa:CP Ligases

SerRS, the N-terminal domain interacts with a conserved HelixTurn-Helix (HTH) motif in the catalytic domain, which reduces the flexibility of the N-terminal domain and orients it for tRNA binding (Bilokapic et al., 2006, 2009). Apart from differently structured N-terminal domains, two additional idiosyncratic features of the methanogenic-type SerRS define a distinct mode of substrate recognition (Bilokapic et al., 2006): the catalytic zinc ion in the active site, which binds serine through its amino group, and the ‘‘serine ordering loop’’ (SOL), a conserved amino acid stretch inserted between class-defining motifs 2 and 3. The SOL undergoes profound induced-fit conformational change upon serine binding, bringing its residues into proximity with the zinc ion, enabling a direct contact between the amino acids in the SOL and serine. Crystal structures have revealed the rearrangement of the SOL from a disordered conformation in the unliganded enzyme to an a-helix followed by a loop (Bilokapic et al., 2006). Our kinetic and thermodynamic experiments (Dulic et al., 2011) confirmed that the observed secondary structure transition is indeed essential for serine activation and aminoacylation because the accommodation of the tRNA within the active site requires repositioning of the SOL (Dulic et al., 2011; Bilokapic et al., 2008). Functional expression of duplicated SerRS genes has been observed in numerous bacteria and in some eukaryotes, increasing the diversity of the SerRS family. Although structurally related, these SerRS-like proteins serve different functions than the housekeeping aaRSs. SerRS forms that were significantly divergent from the canonical bacterial SerRSs were found in two antibiotic-producing Streptomyces species. In S. viridifaciens, the duplicated, pathway-specific SerRS provides seryl-tRNASer to be used as precursors for valanimycin biosynthesis (Garg et al., 2006), while an additional SerRS homolog encoded by the gene within the albomycin biosynthesis cluster in Streptomyces sp. ATCC 700974 confers resistance to its host (Zeng et al., 2009). A SerRS-like protein (named SLIMP) with an essential but undefined mitochondrial function has been identified in insects (Guitart et al., 2010). This SerRS paralog has lost the ability to bind amino acids and ATP but has retained the tRNA binding ability. We have recently described and characterized SerRS-like proteins that share low sequence identity with the catalytic domain of methanogenic-type SerRSs. The homologs activate selected amino acids, are deprived of the tRNA-binding domain, and lack canonical tRNA aminoacylating activity (Mocibob et al., 2010). Remarkably, two Bradyrhizobium japonicum enzymes activate glycine instead of serine, while an Agrobacterium tumefaciens homolog preferentially activates alanine. These activated amino acids are then transferred to a new macromolecular substrate, specific carrier proteins (CPs), which share signature motifs with acyl carrier proteins (ACPs), and peptidyl carrier proteins (PCPs). ACPs and PCPs act as the carriers of activated acyl moieties in fatty acid and secondary metabolite synthesis, respectively (Mercer and Burkart, 2007). CPs associated with SerRS homologs thus represent additional members of the versatile carrier protein family. CPs are posttranslationally modified with a 40 -phosphopantetheine (Ppant) to serve as a prosthetic group and methanogenic-type SerRS homologs, whose genes were identified in numerous bacterial species, to

catalyze thioesterification of the Ppant prosthetic group. Therefore, the homologs act as amino acid:[carrier protein] ligases (aa:CP ligases). aa:CP ligases are dimeric enzymes, like all SerRSs, and their catalytic activity is zinc-dependent as previously determined for methanogenic-type SerRSs. Crystallographic analysis confirmed that aa:CP ligases are indeed structurally very similar to the catalytic core of mMbSerRS (Mocibob et al., 2010); however, they functionally resemble otherwise unrelated adenylation domains (Gulick, 2009) found in nonribosomal peptide synthetases. aa:CP ligases might represent a glimpse into the evolution of class II aaRS that originated from the ancient catalytic core capable of amino acid activation before it gained tRNA acylation activity. According to the crystal structures of Gly:CP ligase 1 from B. japonicum and its complexes with small substrates and substrate analogs (Mocibob et al., 2010), the serine ordering loop (SOL), the conformation of which in mMbSerRS is markedly altered upon serine binding, is replaced by a loop-helix element (LH) in the truncated homolog and does not display substrate induced-fit changes. Here, we present the crystal structures of the complex between B. japonicum Gly:CP ligase 1 (Bj Gly:CP ligase 1) and its cognate carrier protein and elucidate the structural basis for specific protein:protein interactions. We demonstrate the important role of the LH structural motif in macromolecular recognition (Figure 1A). Our biochemical experiments revealed that CPs, encoded by the genes in the genomic surroundings of aa:CP ligase (cognate CPs), act as the specific macromolecular acceptors of amino acids. In contrast to SerRS:tRNA complexes (Figure 1B), CPs do not bind across two subunits of dimeric ligases and their prosthetic group approaches the active site of an amino acid-activating enzyme from a different direction than tRNA through a tunnel unseen in the mMbSerRS structure. Similar to tRNA:aaRS systems, some aa:ligases are quite selective (as B. japonicum ligase). Others (as A. tumefaciens enzyme) are more relaxed in CP recognition. Structural analyses of these macromolecular complexes allow us to postulate that the helix within the LH structural motif is a key determinant of CP binding. Biochemical analyses of several mutants provide compelling support for this hypothesis. The structures also provide a view of the residues that form the protein interface and allowed us to modify the adenylating enzyme and to engineer its specificity to efficiently recognize a heterologous substrate (a noncognate CP). RESULTS Carrier Proteins Recognize Cognate aa:CP Ligase through the Interaction with the Distinctive CP-Binding Helix To reveal the interaction between aa:CP ligase and its cognate carrier protein at the molecular level, we crystallized and solved the structure of Bj Gly:CP ligase 1 complexed with its cognate CP (Bj CP) at 2.15 A˚ resolution (Protein Data Bank [PDB] ID 4H2S). The asymmetric unit contains one molecule of homodimeric Gly:CP ligase and two CPs, each bound to one subunit of the Gly:CP ligase (Figure 1A). Bj Gly:CP ligase 1 in the complex is almost identical to the uncomplexed ligase structure (Ca rootmean-square-deviation [rmsd] 0.49). The structure of Bj CP is very similar to the previously published structures of acyl-carrier

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Structure Carrier Protein Recognition by aa:CP Ligases

Figure 1. The Structure of B. japonicum Gly:CP Ligase 1 in Complex with Cognate CP (A and B) Structure comparison of Bj Gly:CP ligase 1 complexed with Bj CP (A) and the model of mMbSerRS complexed with tRNA (B). The two subunits of the enzymes are colored in gray and yellow. Both enzymes contain Zn2+ in the active site, shown as a cyan sphere. The N-terminal domain (pink), important for recognition of tRNA, does not exist in Bj Gly:CP ligase 1. Macromolecular substrates, CP and tRNA, are shown in teal. One tRNA molecule binds across both subunits of mMbSerRS, while CP binds to a distinct CP-binding helix (light pink) of the ligase. The CP-binding helix is a part of loop-helix motif (light pink) characteristic for aa:CP ligases. In mMbSerRS, the loop-helix is replaced by the serine ordering loop (light pink) that is formed upon the serine binding. The thin ribbon in panel A corresponds to the poorly structured part of the CP untraceable in the electron density maps. See also Figures S1 and S2.

proteins. Bj CP is a small protein composed of 90 amino acids and containing a 40 -phosphopantetheine group covalently attached to a conserved serine residue (Ser42). As with most known CP structures, it has a helical bundle fold containing three major helices and a minor distorted one, with Ser42 located at the beginning of the second major helix. The CP binds to the ligase through interaction with an idiosyncratic solvent-exposed helix, held on a long loop (the loop-helix structural element, LH) (Figures 1A and 2A). Therefore, we refer to this distinctive helix as the CP-binding or CP-recognition helix. The loop and the CP-binding helix correspond to an insertion characteristic of atypical SerRSs and homologous aa:CP ligases. Contrary to mMbSerRS, where this region (SOL) adopts a different conformation upon serine binding (Bilokapic et al., 2006), LH is placed farther away from the active site of Bj Gly:CP ligase 1, and amino acid binding does not induce significant conformational changes. Instead, the CP-binding helix is engaged in CP binding only. In all Gly:CP ligase structures (Mocibob et al., 2010), when uncomplexed with CP, the region belonging to the CP-binding helix (residues A213–Q233) is well ordered only in one subunit of the homodimer due to the contacts involved in crystal packing interactions, while it is highly flexible in the other subunit. This observation suggests that the binding of cognate CP constrains the inherent flexibility of the CP-binding helix. Two CPs interact solely with the Bj Gly:CP ligase 1 and demonstrate no crystal packing constraints, resulting in higher flexibility and higher B-factors, which is reflected in poorly defined parts of the electron density maps corresponding to the carrier proteins (Figure S1 available online). One CP is better ordered and residues 7–80 (out of 90 amino acids) have been successfully built, while for the other CP, only resi-

dues 34–70 have been placed in the poorly defined electron density. The average B-factor for all atoms of Gly:CP ligase is 23.4 A˚2. For the more well-ordered CP, the average B-factor is 78.2 A˚2. The average B-factor is 94.8 A˚2 for the more disordered CP (calculated using B average, part of the CCP4 suite; Winn et al., 2011). The interface, calculated using the PISA server (European Bioinformatics Institute; Krissinel and Henrick, 2007), between Gly:CP ligase and its CP comprises approximately 650 A˚2, representing 13.2% of the available surface area of the CP and 4.5% of the available surface area of the ligase (Figure 2A). The carrier protein binds to the CP-binding helix of the ligase mostly through hydrophobic interactions. Only two Gly:CP ligase residues, Arg220 placed at the N terminus and Gln231 at the C terminus of the CP-binding helix, form two putative hydrogen bonds with the CP at the interaction sites most exposed to the solvent. The Arg220 side chain interacts with the backbone carbonyl oxygen of the Ile60 and Glu53 side chain of the carrier protein. Gln231 forms putative hydrogen bonds with the Pro67 and Phe70 residues of the CP backbone (Figure 2B). The interaction is mainly achieved by extensive hydrophobic interactions between three methionine residues, where Gly:CP ligase Met224 is stacked between Met45 and Met49 of the CP (Figure 2C). This hydrophobic core is surrounded by additional hydrophobic interactions that involve CP Ile65 and ligase Val227 and CP Val46 and ligase Val221. The Ppant Prosthetic Group Enters the Ligase through an Idiosyncratic Tunnel Leading to the Active Site The 40 -phosphopantetheine (Ppant) arm enters deep into the catalytic domain of the ligase into the proximity of the zinc ion and the AMP molecule bound in the active site. While the

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Structure Carrier Protein Recognition by aa:CP Ligases

Figure 2. Protein:Protein Interaction between Bj Gly:CP Ligase 1 and Cognate CP (A) CP (teal) binds solely to the CP-binding helix (light pink) of the ligase (gray). (B) Putative hydrogen bonds formed between CP and Bj Gly:CP ligase 1 are located on solventexposed part of the interface. (C) Hydrophobic interactions involved in CP-ligase interaction. Zinc ion is shown as a cyan sphere. Residues coordinating the zinc ion, AMP, 40 -phosphopantetheine, and residues involved in CP:ligase interaction are shown as sticks. Putative hydrogen bonds are marked as black dashes. Roman numerals designate four characteristic CP helices.

30 -CCA end of tRNA approaches the active site from the motif 2 loop direction, the Ppant chain enters the active site from the opposite direction (Figures 1A and 1B; Figure S2) through a tunnel formed between the LH structural motif and the catalytic body of the ligase. This tunnel is idiosyncratic to aa:CP ligase and does not exist in the structure of mMbSerRS, where it is blocked by the serine ordering loop located close to the amino acid binding pocket. In the structure of mMbSerRS, slightly rearranged helices 8 and 14 and the tip of the outer helix of the HTH motif spanning from the other subunit also fill the space where the tunnel is located in the Bj Gly:CP ligase 1 structure. In addition, the access to such a tunnel in mMbSerRS would be obstructed by the N-terminal tRNA-binding domain, which would completely cover its opening (Figure 1). It is noteworthy that the part of the synthetase catalytic core that interacts with the tRNA 30 -CCA end is among the most structurally conserved regions across the class II aaRS superfamily (O’Donoghue and Luthey-Schulten, 2003). Furthermore, all class II aaRSs bind tRNA in the same orientation in relation to the catalytic core. The mode of tRNA binding is one of the hallmarks that distinguish class I and class II aaRSs (Arnez and Moras, 1997). Therefore, aa:CP ligases have evolved a distinct mode of Ppant chain access and accommodation into the active site. The Gln232 side chain of the ligase stabilizes the phosphate group of the Ppant moiety. There are few putative hydrogen bonds between the ligase and Ppant: the sulfhydryl group and His257, the pantoyl carbonyl oxygen and Gln229 side chain, and the b-alanyl carbonyl oxygen and Tyr132. These hydrogen bonds were not consistently observed in all herein described crystal structures of the complex and they are probably transient. The average B-factor for the prosthetic group is 62.1 A˚2, which is lower than the average B-factor for the CP, indicating that the interaction with the ligase stabilizes the Ppant conformation to a limited extent (Figure S1).

Apparently, the prosthetic group makes no specific contacts with the wall of the tunnel, leaving the Ppant moiety rather flexible and unrestrained compared to the residues of the ligase molecule surrounding the prosthetic group (the average B-factor for the ligase residues is 23.1 A˚2). This might be of functional relevance in terms of providing the prosthetic group the flexibility needed for the reaction to occur. The Proposed Catalytic Mechanism of Amino Acid Transfer to the CP Prosthetic Group To observe how small substrates bind into the active site of Bj Gly:CP ligase 1 in the presence of CP, we soaked crystals of the complex with ATP (Figure S3A) or GlyAMS (50 -O-[N-glycylsulfamoyl]adenosine), which is a glycyl-adenylate analog (Figure 3A). Structures of the CP complexes with small ligands (ATP and GlyAMS) are nearly identical to the initial complex (Ca rmsd 0.48 and 0.40 A˚, respectively). ATP is bound in the active site of the CP complex (PDB ID 4H2U) in the bent conformation in the same manner that ATP binds to uncomplexed Bj Gly:CP ligase (PDB ID 3MEY; Mocibob et al., 2010). The position of GlyAMS is also similar in free Bj Gly:CP ligase 1 (PDB ID 3MF1; Mocibob et al., 2010) and in the ligase complexed with CP (PDB ID 4H2T), with the glycine amino group oriented toward the zinc ion. In the structure of the complex with GlyAMS, the sulfhydryl group of the Ppant arm seems to be oriented toward the carbonyl moiety of the glycine and placed in the correct position for the nucleophilic attack (Figure 3A). In the structures with AMP and ATP, this is not the case. Instead, the Ppant sulfhydryl group seems to be oriented toward the His257 residue (Figure S3A). To elucidate the mechanism of the second step of the reaction, the transfer of activated glycine from glycyl-adenylate (GlyAMP) to the CP prosthetic group, crystals of the complex were soaked with ATP and glycine. After providing all the substrates needed for the reaction, in one of the active sites we observed the density corresponding to the glycine moiety bound on the CP Ppant chain (Figure S3B) (Gly-Ppant; PDB ID 4H2V). Due to the small size of the glycine moiety and the flexible nature of the Ppant group in all Gly:CP ligase structures, the electron density for the Gly-Ppant could be interpreted as Ppant and

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Structure Carrier Protein Recognition by aa:CP Ligases

Figure 3. Active Site of the B. japonicum Gly:CP Ligase Complex (A) Active site of Bj Gly:CP ligase 1 complexed with glycyl-adenylate analog (GlyAMS). The prosthetic arm (Ppant) is positioned toward the carbonyl moiety of GlyAMS. (B) Proposed mechanism for the transfer of activated glycine to Ppant. Prosthetic group and GlyAMS are displayed as yellow sticks and the residues involved in the reaction are shown as gray sticks. Putative hydrogen bonds are marked as black dashes. The red sphere represents a water molecule, while the cyan and green spheres correspond to zinc and magnesium ions, respectively. See also Figure S3.

a water molecule. The calculated omit maps revealed that the Gly-Ppant moiety fits slightly better in the electron density (Figures S3C and S3D). However, it cannot be excluded that Ppant and a water molecule or mixture of both species exist in the crystal. The structures of the macromolecular complex were soaked with different substrates (AMP, ATP, Gly combined with ATP; Figures S3A and S3B) and GlyAMS (Figure 3A) provided us with the different snapshots of the catalytic cycle and allow us to speculate about the reaction mechanism. Our previous work (Mocibob et al., 2010) revealed that the adenylate binding pocket of Bj Gly:CP ligase 1 is highly conserved as in class II aaRS synthetases and thus it is reasonable to assume that the glycine activation step, glycyl-adenylate formation, proceeds through a conventional in-line displacement mechanism by nucleophilic

attack of the glycine carboxylate group to a-phosphate of ATP. The second step of the reaction involves a nucleophilic attack of the Ppant sulfhydryl group on the glycyl-adenylate carbonyl (Figure 3B), leading to thioester bond formation. In the structure of the complex soaked with a glycyl-adenylate analog (PDB ID 4H2T), the Ppant sulfhydryl group is located close to GlyAMS and is favorably oriented to perform a nucleophilic attack on the carbonyl atom of GlyAMS. The sulfhydryl group most likely forms a hydrogen bond with the nonbridging phosphate oxygen of the adenylate, which helps to properly orient the attacking nucleophile and allows uptake of the sulfhydryl proton by the pro-R nonbridging phosphate oxygen of the adenylate. The universally conserved motif 2 arginine (Arg159) present in all aaRS class II enzymes directly contacts carbonyl and pro-R nonbridging adenylate oxygen and most likely contributes to additional polarization of the reacting groups. Lys235 makes a salt bridge with pro-S nonbridging phosphate oxygen and, together with Arg159, participates in the stabilization of the negative charges in the transition state. Thus, the thioester bond formation could proceed through a substrate-assisted concerted mechanism without direct participation of protein residues acting as the general base during amino acid transfer. This proposed mechanism is similar to the mechanism of amino acid transfer to tRNA proposed for AspRS (Eiler et al., 1999) or HisRS (Guth et al., 2005). On the other hand, only three residues of Bj aa:CP ligase 1, Tyr132, Asp215, and His257, were found in sufficient proximity to the reaction center to potentially participate as a general base and subtract the proton from the Ppant sulfhydryl group, but mutagenesis experiments failed to provide support for this alternative reaction mechanism. The effects of Y132F and H257A mutations on the enzyme activity were negligible, while D215A mutation completely inactivated the enzyme. The Asp215 residue makes a salt bridge with the active-site residue Lys235 and is most likely crucial for correct positioning of Lys235, which plays an essential role during amino acid activation. Because the D215A mutation incapacitated amino acid activation, discussion of its role in the subsequent amino acid transfer step is precluded. The K235A mutant is also completely inactive in both amino acid activation and the overall aminoacylation reaction. Both residues are strictly conserved in aa:CP ligase sequences (Figure S4A). Specificity of aa:CP Ligases toward CPs Multiple alignment of representative aa:CP ligase sequences revealed that the loop preceding the CP-binding helix (A213-G219 in Bj Gly:CP ligase 1) in proximity to the active site is evolutionary well conserved, while the sequence of the CP-binding helix (R220-Q232) displays unexpected sequence variability (Figure S4A). Among Bj Gly:CP ligase 1 helix residues in direct interaction with CP, only Arg220, Met224, and Gln232 are weakly conserved. This observation prompted us to perform a more detailed phylogenetic analysis of aa:CP ligases and corresponding CPs (see Supplemental Results). aa:CP ligases and their CPs are scattered unevenly across the bacterial domain and they are predominantly present in the members of Alphaproteobacteria and Betaproteobacteria classes. Phylogenetic trees were constructed with the Neighbor Joining, Maximum Likelihood, and Maximum Parsimony methods (Figure S5). Phylogenetic

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Structure Carrier Protein Recognition by aa:CP Ligases

Table 1. Kinetic Parameters for CP Aminoacylation by B. japonicum, A. tumefaciens, and the Hybrid aa:CP Ligases KM (mM)

kcat (s1) 0.11 ± 0.02

kcat / KM (103 s1 M1)

Bj Gly:CP ligase 1

Bj CP

1.19 ± 0.04

At CP

–

At Ala:CP ligase

At CP

4.7 ± 0.5

0.31 ± 0.01

68 ± 7

Bj CP

47 ± 2

0.34 ± 0.02

7.2 ± 0.3

hybrid Gly:CP ligase

At CP

1.9 ± 0.3

0.119 ± 0.003

64 ± 9

Bj CP

35 ± 3

0.122 ± 0.008

3.6 ± 0.4

–

91 ± 7 1.22 ± 0.03

Due to differences in amino acid specificities, Bj and the hybrid Gly:CP ligase were assayed with glycine, while alanine was used to assay At Ala:CP ligase. The values are given as the arithmetic mean ± SEM.

analysis yielded unanticipated results because the topology of CP phylogenetic trees does not correspond to the topology of aa:CP ligase phylogenetic trees. Most importantly, A. tumefaciens and B. japonicum CPs are located within distantly related clades of the CP phylogenetic tree, although both species belong to the same order Rhizobiales of the class Alphaproteobacteria. Because CP genes are found close to aa:CP ligase genes, they were expected to coevolve. Low conservation of the CP interacting domain in aa:CP ligases and phylogenetic analysis (SI Results and Figure S5), which revealed that A. tumefaciens (At) and B. japonicum (Bj) aa:CP ligases and especially CPs are not so closely related as anticipated, suggested that At and Bj aa:CP ligases might be quite selective in recognition and aminoacylation of their respective CPs. Indeed, kinetic analysis confirmed that At and Bj aa:CP ligases are quite specific for cognate CPs, encoded by the genes located in the vicinity of the ligase genes, and cross-aminoacylate heterologous CPs rather inefficiently. At Ala:CP ligase discriminates cognate and heterologous CP from B. japonicum 10-fold (Table 1), as a result of decreased affinity (increased KM) for heterologous Bj CP, consistent with the premise that differences at the interaction interface would affect CP binding but not the amino acid activation and transfer in the active site. Bj Gly:CP ligase 1 is even more specific and aminoacylates cognate Bj CP with 75-fold higher kcat/KM than heterologous At CP. Due to the low affinity of Bj Gly:CP ligase 1 for At CP, saturation could not be achieved and individual catalytic parameters were not determined. Thus, kinetic analysis confirmed that At and Bj aa:CP ligases inefficiently recognize heterologous CPs. It is noteworthy that aa:CP ligases are even more discriminative toward acyl carrier proteins (ACPs) dedicated to fatty acid synthesis, which they aminoacylate several orders of magnitude less efficiently than cognate CPs (Mocibob et al., 2010), although they share the same fold and properties. The results of the kinetic analysis were further corroborated by isothermal titration calorimetry (ITC) and pull-down experiments. In pull-down experiments, aa:CP ligases were incubated with different His-tagged CPs, and the mixture was loaded on affinity resin, on Ni-NTA agarose. aa:CP ligases are retained on the resin only if they interact with immobilized His-tagged CPs. Bj Gly:CP ligase 1 (Figure 4A) was specifically retained on the affinity resin only in the presence of cognate CP. Heterologous At CP was not a competent bait for Bj Gly:CP ligase 1 retention

on affinity resin. Contrary to our expectation, there was no evident difference in At Ala:CP ligase binding to cognate At CP or heterologous Bj CP in pull-down assays (Figure 4B). However, pull-down assays provide only qualitative binding information, which might be misleading. As kinetic analysis revealed, At Ala:CP ligase is more relaxed than Bj Gly:CP ligase 1 in CP binding and discriminates heterologous Bj CP only 10-fold. The results of the pull-down assay might reflect this relaxed specificity of At Ala:CP ligase. The interaction of aa:CP ligases and CPs was more rigorously and quantitatively studied by ITC. ITC experiments confirmed that At and Bj aa:CP ligases bind only cognate CPs with moderate affinity (Kd = 33.2 and 21.5 mM, respectively). Calorimetric titration of aa:CP ligases with heterologous CPs did not yield measurable enthalpy changes, indicating that heterologous CPs do not bind to an observable extent. Taken together, the results of the kinetic, pull-down, and ITC experiments demonstrate that aa:CP ligases specifically bind cognate CPs. The Importance of the Phosphopantetheine Prosthetic Group for Ligase:CP Complex Formation aa:CP ligases from B. japonicum and A. tumefaciens are specific and dedicated to aminoacylation of cognate CPs. In our previous work (Mocibob et al., 2010), it was shown that aa:CP ligases do not aminoacylate ACPs involved in fatty acid synthesis to a significant extent, although ACPs represent a carrier protein prototype with a similar four-helix bundle fold and identical prosthetic group. Thus, the prosthetic group is not a predominant element in ligase:CP complex formation. Because aa:CP ligases are specific for cognate CPs and efficiently discriminate macromolecular substrates of similar overall fold, the interaction with the protein component of CPs has to be decisive for CP recognition and binding. An analysis of the crystal structure of the complex supports this assumption: the interaction between CP and ligase relies predominantly on protein:protein interactions, while the prosthetic group appears flexible and apparently does not contribute significantly to CP binding (Figure 2). Therefore, we expected that apoCPs (the CPs devoid of the prosthetic group) should efficiently bind to aa:CP ligases. Surprisingly, this was not the case. First, the influence of apoCPs on aminoacylation kinetics was tested. apoCPs were expected to compete with intact holoCPs for enzyme binding. However, no inhibition was observed, even upon addition of apoCPs to the reaction mixture in substantial excess over holoCPs (Figure 5). Apparently, apoCPs are unable to bind to At and Bj aa:CP ligases under the conditions where holoCPs are readily aminoacylated, implying that the prosthetic group does play an important role in CP binding. The absence of interaction between aa:CP ligases and apoCPs was directly confirmed by isothermal calorimetric titration of At and Bj aa:CP ligases with corresponding apoCPs as well as by pulldown experiments. In pull-down experiments, neither Bj Gly:CP ligase 1 (Figure 4A) nor At Ala:CP ligase (Figure 4B) were immobilized on affinity resin when His-tagged apoCPs were used as the baits. Such a prominent influence of the Ppant group on CP binding is not easy to explain. One possibility is that the phosphopantetheinylation of CP markedly affects conformation of the protein and consequently its interactions, as in the case of

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Structure Carrier Protein Recognition by aa:CP Ligases

Figure 4. Pull-Down Assay of Bj Gly:CP Ligase 1 (A–D) At Ala:CP ligase (B), hybrid Gly:CP ligase (C), and Bj Gly:CP ligase with deleted LH motif (D). The ligases were incubated with various cognate, heterologous, and holo- and apo-carrier proteins, as indicated on the picture. The mixture of Histagged CP and the ligase (input; left side of each panel) was loaded on Ni-NTA agarose, and proteins retained on the resin were analyzed by SDS-PAGE (pull-down; right side of each panel). Bold arrows (lig./) mark the position of the ligases, hollow arrows (CP, open right arrow) designate the position of the CPs on the gel. The numbers in the middle of the gels designate the molecular weight of the MW standards. See also Figures S4 and S5.

changes following CP phosphopantetheinylation certainly require further investigation.

peptidyl CP domain of the third module of tyrocidine A synthetase (TycC3-PCP) (Koglin et al., 2006). Therefore, we have recorded and compared circular dichroism (CD) spectra of At and Bj apo- and holoCPs. CD spectra of the apo- and holoforms were virtually identical (Figure S6), with a prominent positive peak at 190 nm and negative peaks at 208 and 222 nm characteristic for a helices. The spectra do not support the presumption of substantial rearrangement of secondary structure elements. Furthermore, after solving the structure of At holoCP in complex with the hybrid ligase (see below), we have superimposed it on a publicly available structure of At apoCP (PDB ID 2JQ4). Superposition of the two structures (Figure S7A) confirmed that the apo- and holo-forms of At CP share a similar overall fold with only minor differences (rmsd = 2.2 A˚). Thus, contrary to TycC3-PCP, the conformation of apo- and holo-CPs appears almost identical, although the minor influence of phosphopantetheinylation on the CP conformation cannot be excluded. Conformation of apo- and holo-ACPs involved in fatty acid synthesis also appears almost identical, with only local perturbations around the Ppant attachment site (Kim et al., 2006; Wu et al., 2009). Nevertheless, it is widely accepted that the inherent flexibility of ACPs is essential for their function (Mercer and Burkart, 2007) and phosphopantetheinylation could affect their structural dynamics. It was shown for the actinorhodin polyketide synthase ACP that although the overall structure of this PKS-ACP is essentially the same following the addition of the Ppant side chain, Ppant attachment led to a 2-fold drop in binding affinity for 40 -phosphopantetheinyl transferase AcpS (Evans et al., 2008). Therefore, it remains an open possibility that prosthetic group attachment affects the structural dynamics of CPs and that aa:CP ligases are capable of sensing these subtle conformational differences between apo- and holo-CPs. The postulated conformational

Construction of the Hybrid aa:CP Ligase As judged from crystal structures of the protein complex, the interaction of aa:CP ligase with CP depends mostly on an idiosyncratic helix located distally to the active site. To further confirm and explore the role of the aforementioned helix on CP recognition, we designed a hybrid protein where CP-binding helix of Bj Gly:CP ligase 1 (R220-Q232) was replaced with the corresponding region from At Ala:CP ligase (Figure S4B). The transplantation of the CP-binding helix from At to Bj Gly:CP ligase resulted in complete inversion of specificity for CP (Table 1; Figure 4C). The hybrid of Bj Gly:CP ligase 1 comprising the At interaction helix preferentially aminoacylates At CP instead of Bj CP (Table 1). The difference in catalytic efficiency (kcat/KM) is 18-fold in favor of heterologous At CP. At CP was a poor substrate for parental Bj Gly:CP ligase 1 and helix transplantation resulted in a 52-fold improvement in catalytic efficiency for At CP aminoacylation. As expected, helix substitution affected only the KM for At and Bj CP, while the turnover numbers (kcat) of the hybrid ligase remained identical to kcat of the parental Bj Gly:CP ligase 1 for the cognate Bj CP. This demonstrates that the binding of CP by the recognition helix and the attachment of amino acid to the Ppant group in the active site proceed in two independent and spatially separated steps, with no effect of the helix substitution on the active site. Because the amino acid specificity is determined by the architecture of the active site inherited from Bj Gly:CP ligase 1, the hybrid enzyme transfers activated glycine to At CP instead of alanine even though At CP is originally charged with alanine by cognate wild-type At Ala:CP ligase. The hybrid ligase is fully functional and transplantation of the helix indispensable for CP binding resulted in complete reversal of CP specificity. Specificity of the hybrid ligase for CP binding was further explored by pull-down assays (Figure 4C) and ITC. In both cases, the interaction of the hybrid ligase was detected only in the presence of At CP, in line with the results of the kinetic analysis. ITC revealed robust interaction of the hybrid with At CP

620 Structure 21, 614–626, April 2, 2013 ª2013 Elsevier Ltd All rights reserved

Structure Carrier Protein Recognition by aa:CP Ligases

Figure 5. Influence of apoCPs on CP Aminoacylation By Bj Gly:CP ligase 1 (left panel) and At Ala:CP ligase (right panel). The concentration of the holoCP was varied, while the concentration of the apoCP was kept constant, as indicated on the graphs, in excess of the holoCP concentration to facilitate the competition between apo- and holoCP forms. See also Figure S6.

(Kd = 1.37 mM), while titration of the hybrid with Bj CP did not result in observable signal changes, indicative of significantly reduced affinity. In line with the presented results, deletion of the interaction helix in Bj Gly:CP ligase 1 resulted in the loss of interaction with either cognate Bj CP or heterologous At CP (Figure 4D). Due to the structural constraints, the loop preceding the interaction helix (S214-G219), which connects the helix to catalytic domain, was also removed. Deletion not only resulted in the complete loss of aminoacylation activity, but also abolished the ability to activate amino acids. This result was expected because Asp215 was removed as the part of deleted loop. As already mentioned, Asp215 is critical for enzyme activity because it interacts with active-site residue Lys235. Structure of the Hybrid Gly:CP Ligase Complexed with At CP To reveal the structural basis of the hybrid Gly:CP ligase specificity for heterologous At CP, the crystal structure of the hybrid ligase complexed with At CP was solved. Furthermore, the interactions in the hybrid ligase complex were expected to closely resemble interactions in At Ala:CP ligase complexed with cognate At CP, providing a deeper insight into the ligase:CP interactions. The crystal structure of the designed hybrid Gly:CP ligase complexed with At CP was solved to 1.95 A˚ resolution (PDB ID 4H2W). A comparison of the hybrid and Bj Gly:CP ligase 1 complexes gives a detailed insight into the CP recognition of aa:CP ligases (Figure 6). As expected, the overall structure of the hybrid Gly:CP ligase in the complex is highly similar to Bj Gly:CP ligase 1 (rmsd 0.39 A˚ across all Ca atoms), including the orientation of the CP-binding helix. The structures of At and Bj CPs in complex with hybrid and Bj ligase are also unexpectedly similar (rmsd 1.1 A˚; Figure S7B), given their low sequence identity (27%) and evolutionary divergence (Figure S5B). In spite of their high structural similarity, the binding mode of the CPs to the hybrid and Bj Gly:CP ligase 1 is markedly different. Using the centers of mass of the CPs (calculated with CALCOM software; Costantini et al., 2008), we calculated that At CP is shifted approximately 9 A˚ toward the Ppant tunnel and rotated by 37 around the engineered CP-binding helix (Figure 6A). Due to the observed shift, At CP is bound closer to the catalytic domain of the hybrid ligase and HTH motif. The position of the serine residue (Ser35) that carries the prosthetic arm is also considerably shifted. However, owing to the flexibility of the serine side chain, the phosphate group of the arm is equally positioned as in Bj CP and the Ppant arm extends toward the active site in a similar manner as in the Bj Gly:CP ligase 1 complex (Figure 6A).

The hybrid ligase-At CP interface reveals a different hydrogen bond pattern with additional polar interactions compared to the Bj Gly:CP ligase 1-Bj CP complex. The Arg220 side chain forms putative hydrogen bonds with the CP Glu46 side chain and the main chain of the CP Phe53 residues (Figure 6B), analogous to the hydrogen bonds observed in the Bj complex. Asn227 forms two putative hydrogen bonds with CP Asp55 and Leu58, while hybrid ligase Asn228 and Gln232 additionally interact with the CP Arg60 residue. These interactions delineate the interface core, which is mostly hydrophobic (Figures 6B–6E). Comparing the surfaces of the ligase CP binding helices (Figures 6F and 6G), it is noticeable that their N terminus is positively charged and identically shaped for both helices due to the conserved Arg220 residue. However, the hydrophobic region in the middle of the helices is differently oriented and shaped. The C terminus of the At CP-binding helix is negatively charged, while the Bj CPbinding helix is mostly hydrophobic. A different hydrophobic surface of the helices along with the charge difference of the helix C terminus enables the two ligases to differentiate various CPs, which is the main reason for different binding modes of the two carrier proteins. Crystal structures of hybrid Gly:CP ligase and At CP with ATP and GlyAMS in the active site (PDB IDs 4H2Y and 4H2X, respectively) reveal small substrates bound in the same manner as in Bj Gly:CP ligase 1. This result confirms that the CP-binding helix has no influence on the binding of small substrates and on the first step of the reaction and strengthens the conclusions about the catalytic mechanisms reached by the analysis of Bj Gly:CP ligase 1 complexes. DISCUSSION The crystal structure of Bj Gly:CP ligase 1 complex revealed that the interaction between the CP and the enzyme relies entirely on the peculiar ligase helix, thereby named the ‘‘CP-binding’’ or the ‘‘CP-recognition helix.’’ Contrary to SOL, its topological counterpart, which plays an active and dynamic role in the mMbSerRS catalytic cycle (Bilokapic et al., 2006; Dulic et al., 2011), the LH element in Bj Gly:CP ligase 1 does not interact with the substrates bound in the active site (Mocibob et al., 2010). Instead, our structural investigation of the Bj Gly:CP ligase 1 in complex with CP identified the CP-binding helix as the principal and sole part of the ligase involved in the interaction with the CP. In contrast to the serine ordering loop in mMbSerRS, the Bj Gly:CP ligase loop-helix does not change conformation upon amino acid binding. Rather, the helix seems to be stabilized through interaction with CP. Subsequent biochemical characterization substantiated with sequence analysis confirmed that the

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Structure Carrier Protein Recognition by aa:CP Ligases

Figure 6. Structural Analysis of aa:CP Ligases’ Specificity toward CP (A) Superimposed complex of Bj Gly:CP ligase 1 (gray) and Bj CP (teal) on the complex of hybrid Gly:CP ligase (violet) and At CP (yellow). The centers of mass for the CPs are shown as black spheres. (B and C) Interface between hybrid Gly:CP ligase (violet) and At CP (yellow). Residues involved in polar (B) and hydrophobic (C) interactions are shown as sticks. Putative hydrogen bonds are marked as black dashes. (D–G)Surface charge representations of Bj CP (D) and At CP (E) oriented to display part of the CP surface interacting with CP binding helices (gray, Bj Gly:CP ligase 1; violet, At helix incorporated in hybrid Gly:CP ligase). Surface charge of Bj (F) and At (G) aa:CP ligase CP-binding helices. The upper parts of the helices correspond to their N termini. See also Figures S7 and S8.

CP-binding helix is indispensable for the interaction with CP and demonstrated that the recognition of CP is specific. aa:CP ligases are not the only example of renegade aaRS homologs that have departed from the conventional role of amino acid attachment to the tRNA 30 -acceptor end in protein biosynthesis. Other examples include glutamyl-queuosine tRNAAsp synthetase (Glu-Q-RS), the GluRS paralog that catalyzes posttranscriptional hypermodification of the tRNAAsp anticodon (Blaise et al., 2005) and PoxA (also known as GenX or YjeA), the paralog of LysRS that mediates posttranslational modification of the elongation factor P (EF-P) (Navarre et al., 2010; Yanagisawa et al., 2010). PoxA attaches (R)-b-lysine to the ε-amino group of Lys34 of EF-P (Roy et al., 2011). Curiously, like aa:CP ligases, both Glu-Q-RS and PoxA are homologous to the catalytic domain of GluRS and LysRS, respectively, and lack the tRNA binding domain. Although Glu-Q-RS and PoxA also transfer amino acids to macromolecular substrates of a different type, their mode of interaction with the macromolecular amino acid acceptor is reminiscent of tRNA binding by homologous aaRS. In the case of Glu-Q-RS, a striking sequence similarity has been observed between the tRNAAsp anticodon stem and the tRNAGlu-acceptor stem (Blaise et al., 2005). Therefore, the tRNAAsp-anticodon stem has been successfully docked on the Glu-Q-RS in a manner analogous to tRNAGlu-acceptor end accommodation by GluRS. EF-P adopts a tRNA-like overall

shape, and Lys34, the site of EF-P b-lysylation, is exposed on the tip of the loop corresponding to the tRNA 30 acceptor end. The crystal structure of the PoxA:EF-P complex was recently solved, revealing that EF-P binds to PoxA in an orientation typical for tRNA binding to class II aaRS (Yanagisawa et al., 2010). Thus, the binding of an amino-acid acceptor to Glu-Q-RS and EF-P mimics binding of tRNA to a homologous aaRS. In the broad sense, by aminoacylating the elongation factor or modifying the tRNAAsp anticodon, PoxA and Glu-Q-RS still indirectly participate in ribosomal protein biosynthesis. In contrast to PoxA and Glu-Q-RS, aa:CP ligases evolved unique and unforeseen mode of macromolecular substrate recognition. CPs bear no resemblance to tRNA, and even the Ppant moiety approaches the activated intermediate in the active site from the opposite direction compared to tRNA 30 -end through an opening idiosyncratic to the aa:CP ligases. In light of such a disparate mode of the macromolecular substrate binding, it is not surprising that fusion of the mMbSerRS tRNA binding domain to Bj and At aa:CP ligases was insufficient to reintroduce tRNA aminoacylation activity in chimeric enzymes (Mocibob et al., 2010). Moreover, the ‘‘main entrance’’ to the active site used by tRNA in class II aaRS appears constricted in Bj Gly:CP ligase 1 and too narrow to accommodate tRNA 30 -end. Amino acids attached to CPs are clearly diverted from ribosomal protein biosynthesis and might participate in nonribosomal peptide synthesis, synthesis of

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Structure Carrier Protein Recognition by aa:CP Ligases

secondary metabolites, signal molecules, cell wall components, posttranslational modifications, or other biological processes. aa:CP ligases functionally closely resemble adenylation domains of modular nonribosomal peptide synthetases (NRPSs) (Gulick, 2009). Most NRPS adenylation domains are integrated in catalytic modules of large multifunctional NRPS and aminoacylate the Ppant arm of the adjacent peptidyl carrier domain. Other adenylation domains are self-standing proteins that aminoacylate peptidyl carrier proteins (PCP) in trans. aa:CP ligases and adenylation domains share the same two-step catalytic strategy. Both families first activate amino acids with ATP to form aminoacyl-adenylate, and then transfer the activated aminoacyl moiety to the Ppant chain of the carrier protein. However, aa:CP ligases and adenylation domains are evolutionary and structurally unrelated, and adenylation domains belong to the so-called ANL superfamily, together with acyl-CoA synthetases and firefly luciferase (Gulick, 2009). The first structure of the adenylation domain EntE in complex with the aryl-carrier protein domain from EntB has recently become available (Sundlov et al., 2012), providing the opportunity to compare CP binding by these functionally equivalent but structurally dissimilar enzymes. The stand-alone adenylation domain EntE and EntB comprising the aryl-CP domain participate in nonribosomal synthesis of siderophore enterobactin in Escherichia coli. The mode of CP recognition by Bj Gly:CP ligase 1 and the EntE adenylation domain is different, both in respect to the CP regions involved in the interaction as well as in the overall manner of CP binding by the enzymes (Figures S8A and S8B). In the Bj Gly:CP ligase 1 complex, the binding helix is encompassed by carrier protein helices II and III (Figure 2). The CP firmly grasps the ligase helix, which possibly allows for some hinge-like movement around the ligase helix axis, as the differences between the structures of the Bj and hybrid ligase complexes might be interpreted (Figure 6A). On the other hand, the EntE adenylation domain interacts with helix II and the long loop 1 connecting CP helices I and II (Figure S8B). The phosphopantetheinylation site is located at the beginning of helix II, in the middle of the interacting region. Adenylation domains are composed of a larger N-terminal subdomain and a smaller C-terminal domain (Figure S8B). The active site is located in the N-terminal domain, in the cleft between the two subdomains. The two subdomains of the EntE adenylation domain form a cradle in which the EntB CP is nested, contrary to Bj and At CPs where CPs embrace the binding helix of the aa:CP ligase. Nevertheless, some analogies can be drawn between these two complexes. In both complexes, the Ppant moiety of CP adopts an extended conformation (Figures S8A and S8B). Like in the Bj Gly:CP ligase 1 complex, the prosthetic group approaches the activated intermediate in the active site of the EntE adenylation domain through a long tunnel. This pantetheine tunnel runs between the N- and C-terminal subdomains of EntE and is created by C-subdomain rotation in the thioester-forming conformation (Sundlov et al., 2012). There are few binding interactions between the Ppant moiety and the encompassing protein tunnel of EntE, so the CP prosthetic group is rather unrestrained, like in the Bj Gly:CP ligase 1 complex. Concurrently with the structure of the EntE complex, the structure of the PA1221 protein from Pseudomonas aeruginosa was solved (Mitchell et al., 2012). PA1221 is a natural two-domain protein composed of adenyla-

tion and PCP domains. Intramolecular interaction of the PCP domain with the adenylation domain is very similar to the interaction between the EntE and EntB aryl-CP domain despite low sequence conservation between the EntE versus PA1221 adenylation domain and the EntB aryl-CP versus PA1221 PCP domain. From the standpoint of the CP, the modality of CP binding to a dedicated CP-recognition helix is also unique. A survey of the available structures of CP complexes has revealed that the interaction of CP with the catalytic partner often relies on CP helices II and III (Figures S8C–S8H). However, none of the structures unveiled a helix equivalent to the CP-recognition helix from aa:CP ligases, which would protrude in-between CP helices II and III, making extensive contacts with these two CP helices. One such example is 40 -phosphopantetheinyl transferase AcpS from Bacillus subtilis (Figure S8C). The interaction of ACP with each subunit of trimeric AcpS is mediated through extensive contacts with ACP helices II and III, but compared to aa:CP ligases, the binding interface is flat with predominantly polar interactions (Parris et al., 2000). Similarly, the interface between ACP and cytochrome P450BioI (Figure S8D) from B. subtilis is rather flat and defined by salt bridges and polar contacts between two proteins (Cryle and Schlichting, 2008). A curious mode of ACP binding involves a D9-stearoyl-ACP desaturase from Ricinus communis (castor) (Guy et al., 2011). Homodimeric desaturase interacts mostly with the ACP helix II and to a lesser extent with the helix III, relying on a long helix that is perpendicular to the ACP helix II (Figure S8E). In a somewhat similar manner, ACP binds to the cytosolic STAS domain from E. coli YchM anion transporter (Figure S8F) (Babu et al., 2010). In the EntF peptidyl carrier protein-thioesterase domain structure (PCP-TE; Figure S8G), the PCP domain is wedged between the globular core of the thioesterase, contacted by CP helix III, and two helices protruding from thioesterase catalytic core that cover the N-terminal part of CP helix II (Frueh et al., 2008). Finally, in the structure of the termination module of the surfactin biosynthetic cluster from B. subtilis (Tanovic et al., 2008), helices II and III of the PCP domain are docked into the groove formed by L-shape-arranged helices aC1 and aC10 of the condensation domain (Figure S8H). In conclusion, the structure of the Bj Gly:CP ligase complex reveals a distinct mode of CP recognition by its interaction partner. Although we have demonstrated the importance of the prosthetic group for CP binding to the aa:CP ligases, the reason remains obscured. We hypothesize that the phosphopantetheinylation might influence CP conformation and/or the structural dynamics, which aa:CP ligases may sense. From the analysis of the crystal structures, no significant contributions of the prosthetic group to CP binding can be pinpointed. The Ppant chain within the tunnel leading to the active site appears flexible with no distinct interactions to constrain its conformation. In line with these conclusions are the results of our previous attempts to use CoA as a surrogate to reveal the binding site of the Ppant chain. Bj1 and At aa:CP ligases can utilize CoA as the amino acid acceptor, albeit less efficiently than CPs (Mocibob et al., 2011). However, in the crystal structure of the Bj Gly:CP ligase 1 in complex with CoA, the coenzyme was bound nonproductively in the active site. The nucleotide part of CoA was found in the adenylate binding pocket and

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Structure Carrier Protein Recognition by aa:CP Ligases

Table 2. Data Collection and Refinement Statistics Bj Gly:CP ligase 1 + Bj CP

Hybrid Gly:CP ligase + At CP

Parameter

AMP

GlyAMS

ATP

Gly+ATP

AMP

GlyAMS

ATP

PDB ID

4H2S

4H2T

4H2U

4H2V

4H2W

4H2X

4H2Y

P212121

P212121

P212121

P212121

P212121

P212121

P212121

92.05, 101.68, 103.98

91.46, 101.22, 104.65

90.71, 100.90, 104.27

90.85, 101.02, 104.41

99.70, 101.25, 103.00

99.57, 101.43, 103.04

99.86, 101.59, 103.42

a, b, g ( )

90.00, 90.00, 90.00

90.00, 90.00, 90.00

90.00, 90.00, 90.00

90.00, 90.00, 90.00

90.00, 90.00, 90.00

90.00, 90.00, 90.00

90.00, 90.00, 90.00

Resolution (A˚)a

46.29–2.15 (2.28–2.15)

46.48–2.45 (2.59–2.44)

46.32–2.10 (2.22–2.10)

46.38–2.00 (2.12–2.00)

45.76–1.95 (2.06–1.95)

45.94–2.15 (2.28–2.15)

46.09–2.10 (2.23–2.10)

Rmeas

8.1 (56.7)

9.6 (63.4)

6.8 (53.3)

6.5 (51.0)

7.3 (52.3)

5.8 (45.7)

7.9 (69.3)

I/sI

22.9 (4.2)

20.8 (3.5)

22.4 (4.4)

22.8 (4.6)

24.3 (5.6)

31.3 (5.1)

18.4 (3.1)

Completeness (%)

99.9 (99.7)

99.7 (98.6)

99.7 (98.4)

99.5 (97.5)

99.3 (96.2)

99.7 (98.5)

99.8 (99.5)

Redundancy

7.4 (7.4)

7.3 (7.3)

7.2 (7.3)

7.4 (7.3)

8.0 (8.0)

12.8 (11.0)

6.1 (6.1)

Data Collection Space group Cell Dimensions a, b, c (A˚)

Refinement Resolution (A˚)

2.15

2.44

2.10

2.00

1.95

2.15

2.10

No. reflections (work/free)

53,840 /2,692

36,621/1,833

56,602/2,833

65,482/3,296

76,469 /3,823

57,625/2,878

62,041/3,101

Rwork/Rfree

0.171/0.207

0.178/0.217

0.175/0.209

0.179/0.210

0.172/0.196

0.167/0.198

0.176/0.210

Protein

5,453

5,270

5,397

5,374

5,840

5,797

5,608

Ligand/ion

92/2

96/12

108/9

110/17

89/3

96/3

104/3

Water

418

215

339

337

370

347

371

No. of Atoms

B-Factors (A˚2) Protein

54.94

53.96

57.10

56.54

49.28

57.17

57.73

Ligand/ion

42.34/23.16

38.31/22.72

45.68/40.33

41.42/30.51

38.25/24.42

47.58/40.48

50.53/43.71

Water

30.86

21.51

35.53

31.21

31.67

39.26

34.70

Bond lengths (A˚)

0.008

0.008

0.007

0.007

0.008

0.008

0.007

Bond angles ( )

1.047

1.128

1.105

1.045

1.074

1.062

1.068

Rmsd

a

The values in parentheses are for the highest-resolution shell.

due to steric constrains, it binds in inverted orientation compared to ATP or a glycyl-AMP analog (Mocibob et al., 2011). In the Bj Gly:CP ligase 1 complexed with CoA, the pantetheine chain is directed out of the active site, and it is completely disordered and could not be traced in the electron density maps. Hence, the interaction of the Ppant moiety and the enzyme is so weak that under conditions of protein crystallization, suboptimal and displaced binding of the CoA nucleotide part dominates over proper pantetheine chain binding. Apparently, not only Bj Gly:CP ligase 1 confuses CoA for ATP. In the crystal structure of medium-chain acyl-CoA synthetase ACSM2A, CoA can also be found nonproductively bound in the ATP binding pocket (Kochan et al., 2009). In conclusion, our structural investigation of Bj Gly:CP ligase 1 complexes with the CP revealed a unique and unforeseen mode of macromolecular interaction, both from the perspective of the aa:CP ligase as well as from the CP perspective. The complexes of Bj and hybrid Gly:CP ligase in comparison to mMbSerRS, and class II aaRSs in general, elucidate the surprising plasticity of the class II aaRS catalytic core in macromolecular amino acid acceptor binding where fundamentally different modes of

macromolecular acceptor accommodation is achieved by slight alterations and adaptations of preexisting structural elements. This work, together with our previously published results (Mocibob et al., 2010), provide a comprehensive structural and biochemical characterization of this enzyme family, closely related to class II aaRSs, with interesting implications for the evolution of class II aaRSs involved in ribosomal protein biosynthesis and curious similarities with the adenylation domains found in nonribosomal peptide synthesis. EXPERIMENTAL PROCEDURES Protein Preparation, Purification, and Crystallization The proteins used in this study were produced as recombinant proteins in E. coli, with or without the His-tag, and were purified by standard procedures on Ni-NTA agarose, or by ion-exchange chromatography. Protein:protein complexes for crystallization were prepared by mixing purified enzymes and corresponding CPs. The crystals of protein complexes were obtained by the hanging drop method. Detailed experimental procedures for protein purification, crystallization, and structure determination are provided in the Supplemental Experimental Procedures. Data collection and refinement statistics are summarized in Table 2.

624 Structure 21, 614–626, April 2, 2013 ª2013 Elsevier Ltd All rights reserved

Structure Carrier Protein Recognition by aa:CP Ligases

Determination of Kinetic Parameters for aa:CP Ligases CP aminoacylation reactions were performed at 30 C in 50 mM Tris/HCl pH 7.5, 150 mM KCl, 0.4 mg/ml BSA, 10 mM MgCl2, 4 mM ATP, 5 U/ml inorganic pyrophosphatase, and varying concentrations of CPs. Reaction mixtures also contained 200 mM [U-14C]-Gly when Bj and the hybrid Gly:CP ligase were assayed or 200 mM [U-14C]-Ala for At Ala:CP ligase assays. The enzyme concentrations were optimized to obtain linear time-courses of product formation. The determination of kinetic parameters was performed as described in Mocibob et al., 2010. Inhibition analysis assays in the presence of apoCPs were performed in a similar manner. The concentration of holoCPs was varied, while the concentration of apoCPs was kept constant above the KM of At or Bj aa:CP ligase for the corresponding holoCP. All determinations of kinetic parameters were repeated at least three times. Pull-Down Assay His-tagged CPs were used as the bait, and untagged aa:CP ligases represented the prey in pull-down experiments. The proteins were mixed together and loaded on Ni-NTA agarose column. Proteins retained on Ni-NTA agarose were eluted with 500 mM imidazole and analyzed by SDS-PAGE. Details of pull-down assay and ITC experiments can be found in the Supplemental Experimental Procedures.

ACCESSION NUMBERS The atomic coordinates and structure factors have been deposited in the PDB. The accession codes are as follows: 4H2S, 4H2T, and 4H2U for Bj Gly:CP ligase 1 and CP complexed with AMP, GlyAMS, and ATP, respectively, bound in the active site. The structure of Bj Gly:CP ligase 1 complexed with glycylated CP is deposited under accession code 4H2V. The accession codes for the hybrid Gly:CP ligase in complex with At CP are 4H2W, 4H2X, and 4H2Y for complexes with AMP, GlyAMS, and ATP, respectively, bound in the active site.

SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Results, eight figures, and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.str.2013.02.017. ACKNOWLEDGMENTS We thank Pavel Afonine for his many useful pieces of advice and help with the structure refinement, especially concerning the refinement of the prosthetic arm. Diffraction data were collected at the European Synchrotron Radiation Facility (ESRF) beamlines BM14 and ID29. We are grateful to all ESRF staff for their assistance, especially to Hassan Belrhali for his valuable support and advice. We are indebted to Gordan Horvat for conducting calorimetric measurements and Silvija Bilokapic for advice and stimulating discussions. We thank Nenad Ban, Silvija Bilokapic, and Vesna Simunovic for critically reading the manuscript. The research leading to this publication has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 283570 (BioStruct-X). This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia (grants 098-1191344-2943 to M.L. and 119-09829131358 to I.W.D.) and Croatian Science Foundation (grant 09.01/293 to I.W.D.). N.I. was a recipient of an EMBO short-term fellowship.

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