Exosite modules guide substrate recognition in the ZiPD/ElaC protein family.

Escherichia coli ZiPD is the best characterized protein encoded by the elaC gene family and is a model for the 3'-pre-tRNA processing endoribonucleases (tRNase Z). A metal ligand-based sequence alignment of ZiPD with metallo-beta-lactamase domain proteins of known crystallographic structure identifies a ZiPD-specific sequence insertion of approximately 50 residues, which we will refer to as the ZiPD exosite. Functionally characterized ZiPD homologs from Bacillus subtilis, Methanococcus janaschii, and human share the presence of the ZiPD exosite, which is also present in the amino-terminal, but not in the carboxyl-terminal, domain of ElaC2 proteins. Another class of functionally characterized tRNase Z enzymes from Thermotoga maritima and Arabidopsis thaliana lack characteristic motifs in the exosite but possess a sequence segment with clustered basic amino acid residues. As an experimental attempt to investigate the function of the exosite we constructed a ZiPD variant that lacks this module (ZiPDDelta). ZiPDDelta has almost wild-type-like catalytic properties for hydrolysis of the small, chromogenic substrate bis(p-nitrophenyl) phosphate. Removal of the ZiPD exosite only affects k(cat), which is reduced by less than 40%, whereas both K' andthe Hill coefficient (measures of the substrate affinity and cooperativity, respectively) remain unchanged. Hence, the exosite is not required for the intrinsic phosphodiesterase activity of ZiPD. Removal of the exosite also does not affect the dimerization properties of ZiPD. In contrast to the wild-type enzyme, ZiPDDelta does not process pre-tRNA, and gel shift assays demonstrate that only the wild-type enzyme, but not ZiPDDelta, binds mature tRNA. These findings show that the exosite is essential for pre-tRNA recognition. In conclusion, we identify a ZiPD exosite that guides physiological substrate recognition in the ZiPD/ElaC protein family.

recognition and processing. Reduced and elongated acceptor stem lengths decrease cleavage efficiency (16) as well as long 5Ј-extensions (14). The tRNase Z from H. volcanii requires the T-arm and the D-arm for pre-tRNA processing, whereas deletion of the anticodon arm had only a small effect on cleavage efficiency (13).
ZiPD shares the metallo-␤-lactamase fold (3) consisting of external ␣-helices that enclose two layers of ␤-sheets. The metallo-␤-lactamase superfamily is continuously expanding and includes numerous hydrolytic enzymes as well as several redox enzymes (17). A common feature of the metallo-␤-lactamase family is the presence of a binuclear metal binding site that is essential for catalysis. The metal binding residues of E. coli ZiPD have only recently been identified (4). In the present study, we used this knowledge for metal ligand-based sequence alignment of E. coli ZiPD with metallo-␤-lactamase domain proteins of known crystallographic structure and identified a specific ZiPD sequence insertion. The characterization of an E. coli ZiPD variant lacking the insertion (ZiPD⌬) shows that this sequence module is required for tRNA binding and pre-tRNA processing, but not for the phosphodiesterase activity with the small chromogenic substrate bis(p-nitrophenyl) phosphate. We conclude that ZiPD proteins possess a specific exosite module that directs physiological substrate recognition. Comparison of this sequence region to functionally characterized tRNase Z proteins gives rise to three subgroups.

EXPERIMENTAL PROCEDURES
Materials-Except when stated otherwise, all fine chemicals were purchased from Sigma. Restriction enzymes were from New England Biolabs (Frankfurt, Germany). Oligonucleotides were synthesized by Genset (Paris, France) and MWG-Biotech (Ebersberg, Germany). DNA sequencing was performed by MWG.
Construction of ZiPD⌬-For construction of the ZiPD deletion mutant, the complete plasmid pETM-ZiPD (3) excluding the coding region for amino acids 153-203 was amplified by PCR using the primers ZiPD152Ϫ (5Ј-TACTAGTTTCTTCAATACGATAGCCATAAC-3Ј) and ZiPD204ϩ (5Ј-AACTAGTGGTAAAGCGCTCGCTATTTTC-3Ј) introducing restriction sites for SpeI (underlined). PCR was performed with KOD Hot Start polymerase (Novagen). The linear PCR product was digested with SpeI and subsequently circularized by ligation. Transformation of E. coli DH5␣ yielded vector pETM-ZiPD⌬. The coding sequence of ZiPD⌬ was verified by DNA sequencing. The resulting plasmid encodes a ZiPD variant with residues 153-203 replaced by Thr-Ser.
Expression and Purification of ZiPDwt and ZiPD⌬-Expression and purification of the amino-terminal His-tagged proteins followed the published procedure (3,4). The molecular mass of the purified protein was determined by mass spectrometry to 31026.4 Da (data not shown), which corresponds nicely to the calculated molecular mass of 31024.3 Da.
Cloning, Expression, and Purification of S. cerevisiae tRNase Z, Trz1-The gene encoding the tRNase Z from S. cerevisiae (YKR079c) was obtained by reverse transcription-PCR using primers Y1Eco (5Ј-TATATAGAATTCATGTTCACATTTATACCCATCACCCATCC-3Ј introducing the restriction site for EcoRI (underlined)) and Y2XStop (5Ј-ATATACTCGAGCTAATTTTTCTTGTGTTTCTTAAGTTTGAC-3Ј introducing the restriction site for XhoI (underlined)) and total RNA isolated from S. cerevisiae. The PCR product was digested with EcoRI and XhoI and cloned into pET32a, yielding pET32a-Trz1. The clone was sequenced to confirm that no mutations were induced by the reverse transcription-PCR. The tRNase Z clone was expressed in Rosetta(DE3)pLysS (Novagen) cells and purified using S-proteinagarose according to the manufacturer's protocol. All tags (S, His, and thioredoxin tags) were removed using recombinant enterokinase (Novagen). Purity of the recombinant protein fractions was checked on 10% SDS-PAGE. As a control, the expression vector pET32a (without insert) was likewise transformed into the strain BL21(DE3), and proteins were isolated and purified as described above. This control purification was performed to show that the tRNase Z activity was not due to any E. coli proteins.
Glutaraldehyde Cross-linking-Proteins (ϳ1 mg/ml) in phosphatebuffered saline were incubated with 0.05% glutaraldehyde for 30 min at room temperature. The reaction was stopped with 1/10 volume 1 M lysine and subsequently analyzed on a reducing SDS-PAGE.
In Vitro Pre-tRNA Processing-In vitro processing was performed with 100 ng of recombinant E. coli ZiPD, 100 ng of ZiPD⌬, or 100 ng of S. cerevisiae tRNase Z (Trz1). Pre-tRNA Tyr from Oenothera berteriana was in vitro transcribed and purified as described elsewhere (18). 2 ng of uniformly labeled pre-tRNA was incubated with 100 ng of protein in 50 mM Tris-HCl, pH 7.1, 5 mM KCl, 5 mM MgCl 2 , 1 M ZnCl 2 for 60 min at 37°C. Samples were immediately extracted with phenol/chloroform to stop the reaction and to remove proteins. Reaction products were analyzed on 8% polyacrylamide gels and identified by autoradiography.

ZiPD Has a Specific Sequence Insertion
Module-Because of a generally low overall sequence similarity, standard multiple sequence alignments of E. coli ZiPD with other members of the metallo-␤-lactamase family are not conclusive and even fail to align the highly conserved metallo-␤-lactamase sequence signature HX(H/E)XD. The recent identification of the ZiPD metal binding residues (4) enabled us to manually refine a multiple sequence alignment of ZiPD with other metallo-␤-lactamase domain proteins of known structure. Positions of metal ligands were considered to be strictly conserved within the protein family and served as anchor points for the improvement of the sequence alignment that compares E. coli ZiPD to human glyoxalase II (19), Desulfovibrio gigas rubredoxin:oxygen oxidoreductase (20), and the metallo-␤-lactamases from Bacillus cereus (21) and Stenotrophomonas maltophilia (formerly Xanthomonas maltophilia) (22) (Fig. 1A). This structure-based sequence alignment identifies a ZiPD-specific sequence insertion module of ϳ50 amino acids, located between the zinc ligands His-141 and Asp-212 (numbering for E. coli ZiPD). Within the metallo-␤-lactamase superfamily, this insertion module is an exclusive feature for ZiPD-like proteins. We will refer to it as the "ZiPD exosite" because this report shows that it functions as an exosite that guides physiological substrate selectivity.
To investigate whether a sequence module similar to the ZiPD exosite is a general feature of the ElaC protein family, we performed a multiple sequence alignment with functionally characterized tRNase Z proteins. We found three subgroups, depending on characteristic sequence signatures at the location of the ZiPD exosite. Very similar sequence segments are found in the ZiPD homologs from B. subtilis and M. janaschii as well as in the human ElaC1 protein. The characteristic feature of the ZiPD-type exosite is a glycineand proline-rich segment (Fig. 1B, GP motif, yellow box). ElaC2 proteins share the ZiPD exosite only in the aminoterminal domain, and it is completely absent in the carboxylterminal domain. The ElaC2-type exosite shares the GP motif and is considerably longer than the ZiPD-type exosite. Interestingly, the third subgroup lacks the GP motif and contains a cluster of 4 -5 basic amino acid residues (Fig. 1B, blue box). This Thermotoga maritima (TM)-type exosite is significantly shorter than the ZiPD-and ElaC2-type exosites.
ZiPD Exosite Is Not Required for Phosphodiesterase Activity and Cooperativity-The phosphodiesterase activity of E. coli ZiPD was first detected with the small, chromogenic substrate bpNPP (3). Later, E. coli ZiPD was also found to have pre-tRNA 3Ј-processing activity (7). The bpNPP kinetic parameters for ZiPD⌬ (insertion sequence replaced by Thr-Ser) are highly similar to the parameters of ZiPDwt (wild-type enzyme) ( Table  I). Deletion of the exosite lowers k cat by less than 40% and has a negligible effect on both KЈ and the Hill coefficient n H . Hence, the ZiPD insertion is not required for phosphodiesterase activity or cooperativity.
ZiPD Exosite Is Not Required for Dimerization-E. coli ZiPD is a dimer in its native state (3). The influence of the ZiPD exosite on the dimerization properties of ZiPD was analyzed by gel filtration and glutaraldehyde cross-linking (Fig. 2, A and B). Recombinant ZiPDwt has a molecular mass of ϳ36 kDa, ZiPD⌬ of ϳ31 kDa/monomer. In analytical gel filtration, ZiPDwt elutes at a volume corresponding to ϳ70 kDa, whereas ZiPD⌬ elutes at a volume corresponding to ϳ60 kDa. Peaks corresponding to the monomers were not detected. In reducing SDS-PAGE, ZiPDwt migrates to ϳ36 kDa and ZiPD⌬ to ϳ31 kDa. Glutaraldehyde cross-linking followed by reducing SDS-PAGE leads to a fraction of ZiPDwt migrating to ϳ70 kDa and a fraction of ZiPD⌬ migrating to ϳ60 kDa. Both analytical gel filtration and glutaraldehyde cross-linking therefore show that ZiPDwt and ZiPD⌬ persist as homodimers in solution. In good accordance, both ZiPDwt and ZiPD⌬ have a 2-fold cooperativity with the substrate bpNPP (Table I).
ZiPD Exosite Is Essential for Pre-tRNA Processing-E. coli ZiPD has 3Ј-pre-tRNA processing endoribonucleolytic activity (7). In this study, we probed 3Ј-pre-tRNA processing activity with pre-tRNA Tyr from O. berteriana, which has been widely used to study tRNA maturation (e.g. Refs. 13, 16, 23). The ElaC2 protein from S. cerevisiae (Trz1) served as a positive control (9). ZiPDwt processes pre-tRNA Tyr in a similar manner to Trz1. Both the mature tRNA Tyr and the removed 3Ј-trailer are detected (Fig. 3). Electrophoretic analysis of the cleavage products reveals that E. coli ZiPDwt and Trz1 cut pre-tRNA Tyr at similar positions. Detection of both the 3Ј-trailer and the mature tRNA prove the specific, endoribonucleolytic activity and rules out a further exoribonucleolytic action. ZiPD⌬ does not process pre-tRNA Tyr (Fig. 3). Neither mature tRNA Tyr nor the removed 3Ј-trailer was detected under experimental conditions that allowed for the processing of a large pre-tRNA Tyr fraction by ZiPDwt and Trz1. In comparison to ZiPDwt, the k cat value of ZiPD⌬ with the substrate bpNPP is decreased by ϳ40%. However, even a similarly decreased activity versus pre-tRNA Tyr is expected to lead to a significant fraction of processed tRNA Tyr under these experimental conditions. In conclusion, ZiPD⌬ lost the pre-tRNA processing properties of ZiPDwt, demonstrating that the ZiPD insertion is essential for pre-tRNA recognition.  ZiPD Exosite Is Essential for tRNA Binding-The cytoplasmic ElaC1 protein from A. thaliana (called nuz) binds mature tRNA (24), and in the case of the native wheat tRNase Z, tRNA affinity chromatography was used for purification (2). In the present study, we used electrophoretic mobility shift assays to probe wheat tRNA binding to Trz1, ZiPDwt, and ZiPD⌬. (Fig.  4). Trz1 has a high affinity for mature tRNA. Upon incubation with Trz1, a large fraction of the wheat tRNA migrated to a higher molecular size than in the absence of Trz1. tRNA binding was also detected for ZiPDwt. However, a larger protein amount (750 ng of ZiPD in comparison to 200 ng of Trz1) was required to detect tRNA mobility shifts; despite this elevated protein level, only a small fraction of the total tRNA bound to ZiPD. This indicates that ZiPDwt has a significantly weaker affinity to this tRNA than Trz1. No tRNA mobility shifts were detected with ZiPD⌬ under identical experimental conditions as with ZiPDwt. Thus, deletion of the ZiPD insertion abolished tRNA binding to ZiPD. The complex of tRNA bound to Trz1 or ZiPDwt migrates to very similar positions despite different molecular sizes of the hook proteins. Trz1, an ElaC2 protein, has a molecular size of ϳ97 kDa, ZiPDwt of ϳ37 kDa, and mature tRNA of ϳ20 kDa. Highly similar migration of the protein-tRNA complex for Trz1 and ZiPDwt is best explained with Trz1 being a monomer in solution and binding one tRNA molecule, whereas in the ZiPD homodimer each monomer binds one tRNA molecule. This model is in agreement with our find- ing that the exosite is essential for tRNA binding because there is only one exosite in the ElaC2 proteins.

DISCUSSION
Despite their prominent role in 3Ј-pre-tRNA processing, substrate selectivity and recognition of the elaC-encoded tRNase Z proteins have only been marginally understood. Here we identified for ZiPD, the ElaC protein from E. coli, an exosite that is essential for pre-tRNA processing and tRNA binding but whose removal affects neither dimerization nor the phosphodiesterase activity toward the small substrate bpNPP.
Almost unaltered bpNPP hydrolytic activity and unaffected dimerization properties of the exosite deletion mutant ZiPD⌬ underline that the overall fold remains intact. In good agreement, the structure-based sequence alignment suggests that the ZiPD exosite is inserted in the metallo-␤-lactamase fold motif without disrupting this general structure, e.g. in the form of an extended loop. Based on the sequence alignment, we mapped the position of the ZiPD exosite exemplary on the structure of the enzyme glyoxalase II (Fig. 5). This model proposes that the exosite is inserted as an extended loop between ␤-sheets 9 and 10, opposite to the binuclear active site. The same picture arises with the other metallo-␤-lactamase domain proteins of known structures. This picture agrees nicely with the biochemical data of the present report, in particular with the barely altered bpNPP hydrolytic activity of ZiPD⌬. Because both ZiPDwt and ZiPD⌬ exist as homodimers in solution, it will be of interest to investigate whether the exosite of one subunit comes into proximity with the active site of the other subunit. The high number of conserved proline and glycine residues in the ZIPD exosite suggests low secondary structure content and a high degree of structural flexibility.
The term exosite is most commonly used for protease substrate determinants that lie outside the active site. Matrix metalloproteases (MMPs) are examples of proteases possessing exosites. Here, the non-catalytic hemopexin domains participate in substrate binding and guide substrate selectivity (25). The ZiPD exosite (ϳ50 residues) is significantly smaller than the MMP hemopexin domains (ϳ200 residues). In addition, the ZiPD exosite appears inside the ZiPD sequence, whereas the MMP exosites are located carboxyl-terminal to the catalytic domain. This work is the first report about exosite features of any metallo-␤-lactamase domain protein. This exceptional characteristic distinguishes ZiPD-like proteins from other metallo-␤-lactamase domain proteins.
The presence of the ZiPD exosite divides the tRNase Z protein family into three different subgroups. The ZiPD-type exosite is present in the functionally characterized ElaC1 proteins from E. coli, B. subtilis, and human. ElaC2 proteins possess a ZiPD exosite-like feature only in the amino-terminal domain, which lacks the catalytic metal binding site. The carboxyl-terminal domain, on the other hand, contains the metal binding site and completely lacks the exosite. There is one report showing that the carboxyl-terminal domain of human ElaC2 is sufficient for 3Ј-pre-tRNA processing (9), which indicates a different mechanism of tRNA recognition. However, a second report by the same group points toward significantly decreased tRNA processing activity of the carboxyl-terminal domain alone (11). Both reports agree that the amino-terminal domain alone does not contain the tRNase Z activity. The ElaC2-type exosite has previously been described as a putative nucleotide binding site due to weak sequence similarity to a P-loop sequence motif (8). Recently, the ElaC2 amino-terminal domain was found to be essential for the so-called RNase65 activity, a term that describes specific endoribonucleolytic cleavage of small target RNAs in the presence of 3Ј-truncated tRNA that lacks several residues at the acceptor stem (11). The target RNA is thought to hybridize with the truncated tRNA, finally forming a pre-tRNA-like complex with a 3Ј-extension. This observation suggests that the amino-terminal ElaC2 exosite is also involved in substrate recognition. A third group of comparably smaller ElaC1 proteins lacks the GP motif and possesses instead a shorter exosite with clustered basic residues. For one member of this TM-type exosite-containing group, the T. maritima tRNase Z, an extraordinary 3Ј-pre-tRNA processing feature was shown. This enzyme processes CCA-containing pre-tRNA and removes the 3Ј-trailer after the CCA triplet instead of cutting after the discriminator (7). The functionally characterized enzyme from A. thaliana, which also possesses the TM-type exosite, removes the 3Ј-trailer after the discriminator (2) but also cleaves off the CCA triplet from mature tRNA (16). This activity was also found for the ZiPD homolog from M. jannaschii, which possesses the characteristic ZiPD-type exosite. It appears that the ZiPD exosite is responsible for overall tRNA recognition, whereas the precise localization of the cleavage site and the antideterminant effect of the CCA motif are guided by other segments of the protein. The organization of the ElaC2 proteins with the exosite only in the amino-terminal domain and the catalytic metal binding site in the carboxyl-terminal domain suggests a similar functional interaction in the ZiPD homodimer. According to this model, the substrate that is recognized by the exosite of one monomer is processed at the metal site of the other monomer.
ZiPD-like proteins act as highly specific endoribonucleases in pre-tRNA maturation. Numerous functional studies on ZiPD homologs from various sources revealed endonucleolytic removal of the 3Ј-trailer from the precursor tRNA while both the mature tRNA and the trailer were left intact. The remarkable substrate and cleavage specificity requires complex substrate recognition, which underlines the physiological significance of the ZiPD exosite in tRNA recognition and substrate selectivity.