Mechanistic insights into the function of 14-3-3 proteins as negative regulators of brassinosteroid signaling in Arabidopsis.

Brassinosteroids (BRs) are vital plant steroid hormones sensed at the cell surface by a membrane signaling complex comprising the receptor kinase BRI1 and a SERK-family co-receptor kinase. Activation of this complex lead to dissociation of the inhibitor protein BKI1 from the receptor and to differential phosphorylation of BZR1/BES1 transcription factors by the glycogen synthase kinase 3 protein BIN2. Many phosphoproteins of the BR signaling pathway, including BRI1, SERKs, BKI1 and BZR1/BES1 can associate with 14-3-3 proteins. In this study, we use quantitative ligand binding assays to define the minimal 14-3-3 binding sites in the N-terminal lobe of the BRI1 kinase domain, in BKI1, and in BZR1 from Arabidopsis thaliana. All three motifs require to be phosphorylated to specifically bind 14-3-3s with mid- to low micromolar affinity. BR signaling components display minimal isoform preference within the 14-3-3 non-ε subgroup. 14-3-3λ and 14-3-3ω isoform complex crystal structures reveal that BKI1 and BZR1 bind as canonical type II 14-3-3 linear motifs. Disruption of key amino acids in the phosphopeptide binding site through mutation impairs the interaction of 14-3-3λ with all three linear motifs. Notably, quadruple loss-of-function mutants from the non-ε group exhibit gain-of-function brassinosteroid signaling phenotypes, suggesting a role for 14-3-3 proteins as overall negative regulators of the BR pathway. Collectively, our work provides further mechanistic and genetic evidence for the regulatory role of 14-3-3 proteins at various stages of the brassinosteroid signaling cascade.

The cytoplasmic synthesis and coupled membrane translocation of eukaryotic polyphosphate by signal-activated VTC complex.

Inorganic polyphosphate (polyP) is an ancient energy metabolite and phosphate store that occurs ubiquitously in all organisms. The vacuolar transporter chaperone (VTC) complex integrates cytosolic polyP synthesis from ATP and polyP membrane translocation into the vacuolar lumen. In yeast and in other eukaryotes, polyP synthesis is regulated by inositol pyrophosphate (PP-InsP) nutrient messengers, directly sensed by the VTC complex. Here, we report the cryo-electron microscopy structure of signal-activated VTC complex at 3.0 Å resolution. Baker's yeast VTC subunits Vtc1, Vtc3, and Vtc4 assemble into a 3:1:1 complex. Fifteen trans-membrane helices form a novel membrane channel enabling the transport of newly synthesized polyP into the vacuolar lumen. PP-InsP binding orients the catalytic polymerase domain at the entrance of the trans-membrane channel, both activating the enzyme and coupling polyP synthesis and membrane translocation. Together with biochemical and cellular studies, our work provides mechanistic insights into the biogenesis of an ancient energy metabolite.

BRASSINOSTEROID INSENSITIVE1 internalization can occur independent of ligand binding.

The brassinosteroid (BR) hormone and its plasma membrane (PM) receptor BR INSENSITIVE1 (BRI1) are one of the best-studied receptor-ligand pairs for understanding the interplay between receptor endocytosis and signaling in plants. BR signaling is mainly determined by the PM pool of BRI1, whereas BRI1 endocytosis ensures signal attenuation. As BRs are ubiquitously distributed in the plant, the tools available to study the BRI1 function without interference from endogenous BRs are limited. Here, we designed a BR binding-deficient Arabidopsis (Arabidopsis thaliana) mutant based on protein sequence-structure analysis and homology modeling of members of the BRI1 family. This tool allowed us to re-examine the BRI1 endocytosis and signal attenuation model. We showed that despite impaired phosphorylation and ubiquitination, BR binding-deficient BRI1 internalizes similarly to the wild type form. Our data indicate that BRI1 internalization relies on different endocytic machineries. In addition, the BR binding-deficient mutant provides opportunities to study non-canonical ligand-independent BRI1 functions.

Structural and functional studies of Arabidopsis thaliana triphosphate tunnel metalloenzymes reveal roles for additional domains.

Triphosphate tunnel metalloenzymes (TTMs) are found in all biological kingdoms and have been characterized in microorganisms and animals. Members of the TTM family have divergent biological functions and act on a range of triphosphorylated substrates (RNA, thiamine triphosphate, and inorganic polyphosphate). TTMs in plants have received considerably less attention and are unique in that some homologs harbor additional domains including a P-loop kinase and transmembrane domain. Here, we report on structural and functional aspects of the multimodular TTM1 and TTM2 of Arabidopsis thaliana. Our tissue and cellular microscopy studies show that both AtTTM1 and AtTTM2 are expressed in actively dividing (meristem) tissue and are tail-anchored proteins at the outer mitochondrial membrane, mediated by the single C-terminal transmembrane domain, supporting earlier studies. In addition, we reveal from crystal structures of AtTTM1 in the presence and absence of a nonhydrolyzable ATP analog a catalytically incompetent TTM tunnel domain tightly interacting with the P-loop kinase domain that is locked in an inactive conformation. Our structural comparison indicates that a helical hairpin may facilitate movement of the TTM domain, thereby activating the kinase. Furthermore, we conducted genetic studies to show that AtTTM2 is important for the developmental transition from the vegetative to the reproductive phase in Arabidopsis, whereas its closest paralog AtTTM1 is not. We demonstrate through rational design of mutations based on the 3D structure that both the P-loop kinase and TTM tunnel modules of AtTTM2 are required for the developmental switch. Together, our results provide insight into the structure and function of plant TTM domains.

Mechanistic insights into the regulation of plant phosphate homeostasis by the rice SPX2 - PHR2 complex.

Phosphate (Pi) starvation response (PHR) transcription factors play key roles in plant Pi homeostasis maintenance. They are negatively regulated by stand-alone SPX proteins, cellular receptors for inositol pyrophosphate (PP-InsP) nutrient messengers. How PP-InsP-bound SPX interacts with PHRs is poorly understood. Here, we report crystal structures of the rice SPX2/InsP6/PHR2 complex and of the PHR2 DNA binding (MYB) domain in complex with target DNA at resolutions of 3.1 Å and 2.7 Å, respectively. In the SPX2/InsP6/PHR2 complex, the signalling-active SPX2 assembles into a domain-swapped dimer conformation and binds two copies of PHR2, targeting both its coiled-coil (CC) oligomerisation domain and MYB domain. Our results reveal that the SPX2 senses PP-InsPs to inactivate PHR2 by establishing severe steric clashes with the PHR2 MYB domain, preventing DNA binding, and by disrupting oligomerisation of the PHR2 CC domain, attenuating promoter binding. Our findings rationalize how PP-InsPs activate SPX receptor proteins to target PHR family transcription factors.

Using Biotinylated myo-Inositol Hexakisphosphate to Investigate Inositol Pyrophosphate-Protein Interactions with Surface-Based Biosensors.

Inositol pyrophosphates (PP-InsPs) are highly phosphorylated molecules that have emerged as central nutrient messengers in eukaryotic organisms. They can bind to structurally diverse target proteins to regulate biological functions, such as protein-protein interactions. PP-InsPs are strongly negatively charged and interact with highly basic surface patches in proteins, making their quantitative biochemical analysis challenging. Here, we present the synthesis of biotinylated myo-inositol hexakisphosphates and their application in surface plasmon resonance and grating-coupled interferometry assays, to enable the rapid identification, validation, and kinetic characterization of InsP- and PP-InsP-protein interactions.

Signatures of antagonistic pleiotropy in a bacterial flagellin epitope.

Immune systems respond to "non-self" molecules termed microbe-associated molecular patterns (MAMPs). Microbial genes encoding MAMPs have adaptive functions and are thus evolutionarily conserved. In the presence of a host, these genes are maladaptive and drive antagonistic pleiotropy (AP) because they promote microbe elimination by activating immune responses. The role AP plays in balancing the functionality of MAMP-coding genes against their immunogenicity is unknown. To address this, we focused on an epitope of flagellin that triggers antibacterial immunity in plants. Flagellin is conserved because it enables motility. Here, we decode the immunogenic and motility profiles of this flagellin epitope and determine the spectrum of amino acid mutations that drives AP. We discover two synthetic mutational tracks that undermine the detection activities of a plant flagellin receptor. These tracks generate epitopes with either antagonist or weaker agonist activities. Finally, we find signatures of these tracks layered atop each other in natural Pseudomonads.

A constitutively monomeric UVR8 photoreceptor confers enhanced UV-B photomorphogenesis.

The plant ultraviolet-B (UV-B) photoreceptor UVR8 plays an important role in UV-B acclimation and survival. UV-B absorption by homodimeric UVR8 induces its monomerization and interaction with the E3 ubiquitin ligase COP1, leading ultimately to gene expression changes. UVR8 is inactivated through redimerization, facilitated by RUP1 and RUP2. Here, we describe a semidominant, hyperactive allele, namely uvr8-17D, that harbors a glycine-101 to serine mutation. UVR8G101S overexpression led to weak constitutive photomorphogenesis and extreme UV-B responsiveness. UVR8G101S was observed to be predominantly monomeric in vivo and, once activated by UV-B, was not efficiently inactivated. Analysis of a UVR8 crystal structure containing the G101S mutation revealed the distortion of a loop region normally involved in stabilization of the UVR8 homodimer. Plants expressing a UVR8 variant combining G101S with the previously described W285A mutation exhibited robust constitutive photomorphogenesis. This work provides further insight into UVR8 activation and inactivation mechanisms and describes a genetic tool for the manipulation of photomorphogenic responses.

Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis.

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8-SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors.

Adjustment of the PIF7-HFR1 transcriptional module activity controls plant shade adaptation.

Shade caused by the proximity of neighboring vegetation triggers a set of acclimation responses to either avoid or tolerate shade. Comparative analyses between the shade-avoider Arabidopsis thaliana and the shade-tolerant Cardamine hirsuta revealed a role for the atypical basic-helix-loop-helix LONG HYPOCOTYL IN FR 1 (HFR1) in maintaining the shade tolerance in C. hirsuta, inhibiting hypocotyl elongation in shade and constraining expression profile of shade-induced genes. We showed that C. hirsuta HFR1 protein is more stable than its A. thaliana counterpart, likely due to its lower binding affinity to CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), contributing to enhance its biological activity. The enhanced HFR1 total activity is accompanied by an attenuated PHYTOCHROME INTERACTING FACTOR (PIF) activity in C. hirsuta. As a result, the PIF-HFR1 module is differently balanced, causing a reduced PIF activity and attenuating other PIF-mediated responses such as warm temperature-induced hypocotyl elongation (thermomorphogenesis) and dark-induced senescence. By this mechanism and that of the already-known of phytochrome A photoreceptor, plants might ensure to properly adapt and thrive in habitats with disparate light amounts.

BAM1/2 receptor kinase signaling drives CLE peptide-mediated formative cell divisions in Arabidopsis roots.

Cell division is often regulated by extracellular signaling networks to ensure correct patterning during development. In Arabidopsis, the SHORT-ROOT (SHR)/SCARECROW (SCR) transcription factor dimer activates CYCLIND6;1 (CYCD6;1) to drive formative divisions during root ground tissue development. Here, we show plasma-membrane-localized BARELY ANY MERISTEM1/2 (BAM1/2) family receptor kinases are required for SHR-dependent formative divisions and CYCD6;1 expression, but not SHR-dependent ground tissue specification. Root-enriched CLE ligands bind the BAM1 extracellular domain and are necessary and sufficient to activate SHR-mediated divisions and CYCD6;1 expression. Correspondingly, BAM-CLE signaling contributes to the restriction of formative divisions to the distal root region. Additionally, genetic analysis reveals that BAM-CLE and SHR converge to regulate additional cell divisions outside of the ground tissues. Our work identifies an extracellular signaling pathway regulating formative root divisions and provides a framework to explore this pathway in patterning and evolution.

Constitutive Activation of Leucine-Rich Repeat Receptor Kinase Signaling Pathways by BAK1-INTERACTING RECEPTOR-LIKE KINASE3 Chimera.

Receptor kinases with extracellular leucine-rich repeat domains (LRR-RKs) form the largest group of membrane signaling proteins in plants. LRR-RKs can sense small molecule, peptide, or protein ligands and may be activated by ligand-induced interaction with a shape complementary SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptor kinase. We have previously shown that SERKs can also form constitutive, ligand-independent complexes with the LRR ectodomains of BAK1-INTERACTING RECEPTOR-LIKE KINASE3 (BIR3) receptor pseudokinases, negative regulators of LRR-RK signaling. Here, we report that receptor chimera in which the extracellular LRR domain of BIR3 is fused to the cytoplasmic kinase domains of the SERK-dependent LRR-RKs BRASSINOSTEROID INSENSITIVE1, HAESA and ERECTA form tight complexes with endogenous SERK coreceptors in the absence of ligand stimulus. Expression of these chimeras under the control of the endogenous promoter of the respective LRR-RK leads to strong gain-of-function brassinosteroid, floral abscission, and stomatal patterning phenotypes, respectively. Importantly, a BIR3-GASSHO1 (GSO1)/SCHENGEN3 (SGN3) chimera can partially complement sgn3 Casparian strip formation phenotypes, suggesting that SERK proteins also mediate GSO1/SGN3 receptor activation. Collectively, our protein engineering approach may be used to elucidate the physiological functions of orphan LRR-RKs and to identify their receptor activation mechanism in single transgenic lines.

Molecular mechanism for the recognition of sequence-divergent CIF peptides by the plant receptor kinases GSO1/SGN3 and GSO2.

Plants use leucine-rich repeat receptor kinases (LRR-RKs) to sense sequence diverse peptide hormones at the cell surface. A 3.0-Å crystal structure of the LRR-RK GSO1/SGN3 regulating Casparian strip formation in the endodermis reveals a large spiral-shaped ectodomain. The domain provides a binding platform for 21 amino acid CIF peptide ligands, which are tyrosine sulfated by the tyrosylprotein sulfotransferase TPST/SGN2. GSO1/SGN3 harbors a binding pocket for sulfotyrosine and makes extended backbone interactions with CIF2. Quantitative biochemical comparisons reveal that GSO1/SGN3-CIF2 represents one of the strongest receptor-ligand pairs known in plants. Multiple missense mutations are required to block CIF2 binding in vitro and GSO1/SGN3 function in vivo. Using structure-guided sequence analysis we uncover previously uncharacterized CIF peptides conserved among higher plants. Quantitative binding assays with known and novel CIFs suggest that the homologous LRR-RKs GSO1/SGN3 and GSO2 have evolved unique peptide binding properties to control different developmental processes. A quantitative biochemical interaction screen, a CIF peptide antagonist and genetic analyses together implicate SERK proteins as essential coreceptor kinases required for GSO1/SGN3 and GSO2 receptor activation. Our work provides a mechanistic framework for the recognition of sequence-divergent peptide hormones in plants.

Identity and functions of inorganic and inositol polyphosphates in plants.

Inorganic polyphosphates (polyPs) and inositol pyrophosphates (PP-InsPs) form important stores of inorganic phosphate and can act as energy metabolites and signaling molecules. Here we review our current understanding of polyP and inositol phosphate (InsP) metabolism and physiology in plants. We outline methods for polyP and InsP detection, discuss the known plant enzymes involved in their synthesis and breakdown, and summarize the potential physiological and signaling functions for these enigmatic molecules in plants.

A genetically validated approach for detecting inorganic polyphosphates in plants.

Inorganic polyphosphates (polyPs) are linear polymers of orthophosphate units linked by phosphoanhydride bonds. Polyphosphates represent important stores of phosphate and energy, and are abundant in many pro- and eukaryotic organisms. In plants, the existence of polyPs has been established using microscopy and biochemical extraction methods that are now known to produce artifacts. Here we use a polyP-specific dye and a polyP-binding domain to detect polyPs in plant and algal cells. To develop the staining protocol, we induced polyP granules in Nicotiana benthamiana and Arabidopsis cells by heterologous expression of Escherichia coli polyphosphate kinase 1 (PPK1). Over-expression of PPK1 but not of a catalytically impaired version of the enzyme leads to severe growth phenotypes, suggesting that ATP-dependent synthesis and accumulation of polyPs in the plant cytosol is toxic. We next crossed stable PPK1-expressing Arabidopsis lines with plants expressing the polyP-binding domain of E. coli exopolyphosphatase (PPX1c), which co-localized with PPK1-generated polyP granules. These granules were stained by the polyP-specific dye JC-D7 and appeared as electron-dense structures in transmission electron microscopy sections. Using the polyP staining protocol derived from these experiments, we screened for polyP stores in different organs and tissues of both mono- and dicotyledonous plants. While we could not detect polyP granules in higher plants, we could visualize the polyP-rich acidocalcisomes in the green alga Chlamydomonas reinhardtii.

Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis.

Many eukaryotic proteins regulating phosphate (Pi) homeostasis contain SPX domains that are receptors for inositol pyrophosphates (PP-InsP), suggesting that PP-InsPs may regulate Pi homeostasis. Here we report that deletion of two diphosphoinositol pentakisphosphate kinases VIH1/2 impairs plant growth and leads to constitutive Pi starvation responses. Deletion of phosphate starvation response transcription factors partially rescues vih1 vih2 mutant phenotypes, placing diphosphoinositol pentakisphosphate kinases in plant Pi signal transduction cascades. VIH1/2 are bifunctional enzymes able to generate and break-down PP-InsPs. Mutations in the kinase active site lead to increased Pi levels and constitutive Pi starvation responses. ATP levels change significantly in different Pi growth conditions. ATP-Mg2+ concentrations shift the relative kinase and phosphatase activities of diphosphoinositol pentakisphosphate kinases in vitro. Pi inhibits the phosphatase activity of the enzyme. Thus, VIH1 and VIH2 relay changes in cellular ATP and Pi concentrations to changes in PP-InsP levels, allowing plants to maintain sufficient Pi levels.

Plant photoreceptors and their signaling components compete for COP1 binding via VP peptide motifs.

Plants sense different parts of the sun's light spectrum using distinct photoreceptors, which signal through the E3 ubiquitin ligase COP1. Here, we analyze why many COP1-interacting transcription factors and photoreceptors harbor sequence-divergent Val-Pro (VP) motifs that bind COP1 with different binding affinities. Crystal structures of the VP motifs of the UV-B photoreceptor UVR8 and the transcription factor HY5 in complex with COP1, quantitative binding assays, and reverse genetic experiments together suggest that UVR8 and HY5 compete for COP1. Photoactivation of UVR8 leads to high-affinity cooperative binding of its VP motif and its photosensing core to COP1, preventing COP1 binding to its substrate HY5. UVR8-VP motif chimeras suggest that UV-B signaling specificity resides in the UVR8 photoreceptor core. Different COP1-VP peptide motif complexes highlight sequence fingerprints required for COP1 targeting. The blue-light photoreceptors CRY1 and CRY2 also compete with transcription factors for COP1 binding using similar VP motifs. Thus, our work reveals that different photoreceptors and their signaling components compete for COP1 via a conserved mechanism to control different light signaling cascades.

Molecular characterization of CHAD domains as inorganic polyphosphate-binding modules.

Inorganic polyphosphates (polyPs) are linear polymers of orthophosphate units linked by phosphoanhydride bonds. Here, we report that bacterial, archaeal, and eukaryotic conserved histidine α-helical (CHAD) domains are specific polyP-binding modules. Crystal structures reveal that CHAD domains are formed by two four-helix bundles, giving rise to a central pore surrounded by conserved basic surface patches. Different CHAD domains bind polyPs with dissociation constants ranging from the nano- to mid-micromolar range, but not nucleic acids. A CHAD-polyP complex structure reveals the phosphate polymer binding across the central pore and along the two basic patches. Mutational analysis of CHAD-polyP interface residues validates the complex structure. The presence of a CHAD domain in the polyPase ygiF enhances its enzymatic activity. The only known CHAD protein from the plant Ricinus communis localizes to the nucleus/nucleolus when expressed in Arabidopsis and tobacco, suggesting that plants may harbor polyPs in these compartments. We propose that CHAD domains may be used to engineer the properties of polyP-metabolizing enzymes and to specifically localize polyP stores in eukaryotic cells and tissues.

Crystal structure of the leucine-rich repeat ectodomain of the plant immune receptor kinase SOBIR1.

Plant-unique membrane receptor kinases with leucine-rich repeat (LRR) extracellular domains are key regulators of development and immune responses. Here, the 1.55 Šresolution crystal structure of the immune receptor kinase SOBIR1 from Arabidopsis is presented. The ectodomain structure reveals the presence of five LRRs sandwiched between noncanonical capping domains. The disulfide-bond-stabilized N-terminal cap harbours an unusual ß-hairpin structure. The C-terminal cap features a highly positively charged linear motif which was found to be largely disordered in this structure. Size-exclusion chromatography and right-angle light-scattering experiments suggest that SOBIR1 is a monomer in solution. The protruding ß-hairpin, a set of highly conserved basic residues at the inner surface of the SOBIR LRR domain and the presence of a genetic missense allele in LRR2 together suggest that the SOBIR1 ectodomain may mediate protein-protein interaction in plant immune signalling.

Crystal structure of the pseudoenzyme PDX1.2 in complex with its cognate enzyme PDX1.3: a total eclipse.

Pseudoenzymes have burst into the limelight recently as they provide another dimension to regulation of cellular protein activity. In the eudicot plant lineage, the pseudoenzyme PDX1.2 and its cognate enzyme PDX1.3 interact to regulate vitamin B6 biosynthesis. This partnership is important for plant fitness during environmental stress, in particular heat stress. PDX1.2 increases the catalytic activity of PDX1.3, with an overall increase in vitamin B6 biosynthesis. However, the mechanism by which this is achieved is not known. In this study, the Arabidopsis thaliana PDX1.2-PDX1.3 complex was crystallized in the absence and presence of ligands, and attempts were made to solve the X-ray structures. Three PDX1.2-PDX1.3 complex structures are presented: the PDX1.2-PDX1.3 complex as isolated, PDX1.2-PDX1.3-intermediate (in the presence of substrates) and a catalytically inactive complex, PDX1.2-PDX1.3-K97A. Data were also collected from a crystal of a selenomethionine-substituted complex, PDX1.2-PDX1.3-SeMet. In all cases the protein complexes assemble as dodecamers, similar to the recently reported individual PDX1.3 homomer. Intriguingly, the crystals of the protein complex are statistically disordered owing to the high degree of structural similarity of the individual PDX1 proteins, such that the resulting configuration is a composite of both proteins. Despite the differential methionine content, selenomethionine substitution of the PDX1.2-PDX1.3 complex did not resolve the problem. Furthermore, a comparison of the catalytically competent complex with a noncatalytic complex did not facilitate the resolution of the individual proteins. Interestingly, another catalytic lysine in PDX1.3 (Lys165) that pivots between the two active sites in PDX1 (P1 and P2), and the corresponding glutamine (Gln169) in PDX1.2, point towards P1, which is distinctive to the initial priming for catalytic action. This state was previously only observed upon trapping PDX1.3 in a catalytically operational state, as Lys165 points towards P2 in the resting state. Overall, the study shows that the integration of PDX1.2 into a heteromeric dodecamer assembly with PDX1.3 does not cause a major structural deviation from the overall architecture of the homomeric complex. Nonetheless, the structure of the PDX1.2-PDX1.3 complex highlights enhanced flexibility in key catalytic regions for the initial steps of vitamin B6 biosynthesis. This report highlights what may be an intrinsic limitation of X-ray crystallography in the structural investigation of pseudoenzymes.