Chlorophyte aspartyl aminopeptidases: Ancient origins, expanded families, new locations, and secondary functions

<div><p>M18 aspartyl aminopeptidases (DAPs) are well characterized in microbes and animals with likely functions in peptide processing and vesicle trafficking. In contrast, there is a dearth of knowledge on plant aminopeptidases with a preference for proteins and peptides with N-terminal acidic residues. During evolution of the Plantae, there was an expansion and diversification of the M18 DAPs. After divergence of the ancestral green algae from red and glaucophyte algae, a duplication yielded the <i>DAP1</i> and <i>DAP2</i> lineages. Subsequently <i>DAP1</i> genes were lost in chlorophyte algae. A duplication of <i>DAP2</i>-related genes occurred early in green plant evolution. <i>DAP2</i> genes were retained in land plants and picoeukaryotic algae and lost in green algae. In contrast, <i>DAP2</i>-like genes persisted in picoeukaryotic and green algae, while this lineage was lost in land plants. Consistent with this evolutionary path, <i>Arabidopsis thaliana</i> has two <i>DAP</i> gene lineages (<i>AtDAP1</i> and <i>AtDAP2)</i>. Similar to animal and yeast DAPs, AtDAP1 is localized to the cytosol or vacuole; while AtDAP2 harbors an N-terminal transit peptide and is chloroplast localized. His₆-DAP1 and His₆-DAP2 expressed in <i>Escherichia coli</i> were enzymatically active and dodecameric with masses exceeding 600 kDa. His₆-DAP1 and His₆-DAP2 preferentially hydrolyzed Asp-<i>p</i>-nitroanilide and Glu-<i>p</i>-nitroanilide. AtDAPs are highly conserved metallopeptidases activated by MnCl₂ and inhibited by ZnCl₂ and divalent ion chelators. The protease inhibitor PMSF inhibited and DTT stimulated both His₆-DAP1 and His₆-DAP2 activities suggesting a role for thiols in the AtDAP catalytic mechanism. The enzymes had distinct pH and temperature optima, as well as distinct kinetic parameters. Both enzymes had high catalytic efficiencies (<i>k</i>_cat/<i>K</i>_m) exceeding 1.0 x 10⁷ M^-1 sec^-1. Using established molecular chaperone assays, AtDAP1 and AtDAP2 prevented thermal denaturation. AtDAP1 also prevented protein aggregation and promoted protein refolding. Collectively, these data indicate that plant DAPs have a complex evolutionary history and have evolved new biochemical features that may enable their role in vivo.</p></div

Kinetic parameters for hydrolysis of Asp-pNA by His₆-DAP1 and His₆-DAP2^A.

<p>Kinetic parameters for hydrolysis of Asp-pNA by His₆-DAP1 and His₆-DAP2^A.</p

The effect of divalent cations on His₆-DAP1 and His₆-DAP2 activity.

<p>The effect of divalent cations on His₆-DAP1 and His₆-DAP2 activity.</p

DAP alignments and phylogenetic relationships.

<p><i>A</i>, Alignments of the <i>Arabidopsis</i> DAP1 (At5g60160) and DAP2 (At5g04710), <i>Oryza sativa</i> DAP1.1 (Os12g13390.1) and DAP2 (Os01g73680), <i>P</i>. <i>falciparum</i> 3D7 M18AAP, <i>Homo sapiens</i> DNPEP, and <i>Bos taurus</i> DNPEP proteins are shown. Accession numbers are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185492#pone.0185492.s003" target="_blank">S3 Table</a>. The two regions that form the globular proteolytic domain based on X-ray crystal structures of the human DNPEP, bovine DNPEP and <i>P</i>. <i>falciparum</i> PfM18AAP are indicated by the heavy grey lines above the protein sequences [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185492#pone.0185492.ref014" target="_blank">14</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185492#pone.0185492.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185492#pone.0185492.ref019" target="_blank">19</a>]. The dimerization domain is located between the proteolytic domains and includes the flexible loop, which contains the His residue that inserts into the catalytic site of its adjacent subunit (dashed grey line) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185492#pone.0185492.s001" target="_blank">S1 Table</a>). His residues that alter DNPEP activity based on biochemical studies and/or X-ray data are shown in red [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185492#pone.0185492.ref013" target="_blank">13</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185492#pone.0185492.ref014" target="_blank">14</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185492#pone.0185492.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185492#pone.0185492.ref019" target="_blank">19</a>]. Residues that have a role in metal coordination, substrate binding/catalysis or that line the catalytic pocket based on one or more X-ray structures are shown in teal. Residues numbers for the human DNPEP, bovine DNPEP and PfM18AAP are for the total protein and differ from residue numbers in the crystal structure determination [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185492#pone.0185492.ref019" target="_blank">19</a>]; these correlations are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185492#pone.0185492.s001" target="_blank">S1 Table</a>. Conserved amino acids (Cons) are indicated and identical residues in the seven DAPs are indicated with a *. <i>B</i>, Phylogenetic relationships of chlorophyte DAPs. Chlorophyte DAPs include DAPs from <i>Arabidopsis thaliana</i> (At), <i>Brachypodium distachyon</i> (Bd), <i>Chlamydomonas reinhardtii</i> (Cr) <i>Chlorella variabilis</i> (Cv), <i>Coccomyxa subellipsoidea</i> (Cs), <i>Cyanophora paradoxa</i> (Cp), <i>Glycine max</i> (Gm), <i>Medicago truncatula</i> (Mt), <i>Micromonas pusilla</i> (Mp), <i>Oryza sativa</i> japonica (Osj); <i>Ostreococcus lucimarinus</i> (Ol), <i>O</i>. <i>tauri</i> (Ot), <i>Physcomitrella patens</i> (Pp), <i>Populus trichocarpa</i> (Pt), <i>Porphyridium cruentum</i> (Pc), <i>Picea sitchensis</i> (Ps), <i>Selaginella moellendorfii</i> (Sm), <i>Sorghum bicolor</i> (Sb), and <i>Vitis vinifera</i> (Vv). <i>Homo sapiens</i> (HsDNPEP), <i>Mus musculus</i> (MmDNPEP), <i>Phytothphora infestans</i> (PiDAP), <i>Aspergillus oryzae</i> (AoDAP), <i>Saccharomyces pombe</i> (SpDAP) and <i>Saccharomyces cerevisiae</i> (ScApe4; ScDAP) DAPs served as outgroups. Accession numbers for all DAP proteins are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185492#pone.0185492.s003" target="_blank">S3 Table</a>.</p

Biochemical characterization of His₆-DAP1 and His₆-DAP2.

<p><i>A</i>, The effects of pH on the activities of His₆-DAP1 (●) and His₆-DAP2 (■) were determined using the Ellis and Morrison buffer system at 37°C using Asp-<i>p</i>NA as a substrate. <i>B</i>, The optimum temperatures for the activities of His₆-DAP1 (●) and His₆-DAP2 (■) were determined at 10°C intervals in the range of 10–90°C using Asp-<i>p</i>NA as a substrate at pH 8.0.</p

His₆-DAP1 and His₆-DAP2 activities after treatment with peptidase inhibitors.

<p>His₆-DAP1 and His₆-DAP2 activities after treatment with peptidase inhibitors.</p

A phosphorylated Ser residue AtDAP1 is imbedded in a Ser-rich region.

<p>The DAP1s and DAP2s from plants and moss, and DAPs from fungi, animals and <i>Plasmodium</i> were aligned by T-coffee and the region from AtDAP1 residues 243 to 336 is displayed. Ser residues are highlighted in gray. Ser284 is phosphorylated in <i>AtDAP1</i> and two predicted phosphorylation sites Ser282 and Ser312 are also shown (♦). AtDAP1’s Thr194 is also predicted as a phosphorylation site (region is not shown). The <i>Plasmodium</i> PfM18AAP has a 17-residue insertion (NTNHTNNITNDINDNIH) in this region.</p

Substrate specificities of His₆-DAP1 and His₆-DAP2.

<p>Substrate specificities of His₆-DAP1 and His₆-DAP2.</p

AtDAP1 and AtDAP2 chaperone activity.

<p>A, Thermal protection assay. NdeI (1 U) was incubated in the presence or absence of His₆-DAP1 (0.2–1.2 μM), His₆-DAP2 (0.1–48 μM; <i>A</i>) or His₆-LAP-A (0.2–2 μM) for 90 min at 43°C. At this time, 140 ng of plasmid DNA was added and digested for 90 min at 37°C. Control lanes show plasmid DNA only and DNA after digestion with unheated NdeI. NdeI cuts at two sites releasing fragments 4.6 kb and 0.2 kb; only the 4.6-kb fragment is shown on these gels. The monomeric supercoiled plasmid (SC) and multimeric supercoils are observed in undigested DNA samples. <i>B</i>, Thermal aggregation assay. CS (300 nM) was incubated with His₆-DAP1 (600 nM; ▲), His₆-DAP2 (300 nM; x), or His₆-LAP-A (900 nM; ■) or without chaperone (♦) at 43°C for 60 min. These concentrations corresponded to CS: DAP1, CS: DAP2 and CS:LAP-A ratios of 2:3, 1:1, and 1:3, respectively. Neither His₆-DAP1 (✳) nor His₆-DAP2 (●) aggregated on their own after heating. Aggregation of CS was determined by measuring light scattering at 360 nm. <i>C</i>, Luc refolding assay. Luc (1 μM) was heated for 11 min at 42°C with 1.5 μM His₆-DAP1 (x), 1.5 μM His₆-DAP2 (○), or 2.2 μM His₆-LAP-A (▲), or no chaperone (■). Luc was allowed to refold in the presence of rabbit reticulocyte lysate (RRL) supplemented with 2 mM ATP. Percent activity corresponds to the relative luminescence compared to unheated luciferase. Measurements were taken for three technical replicates. Data for all panels are representative of two or more independent experiments.</p

A Rationally Designed Agonist Defines Subfamily IIIA Abscisic Acid Receptors As Critical Targets for Manipulating Transpiration

Increasing drought and diminishing freshwater supplies have stimulated interest in developing small molecules that can be used to control transpiration. Receptors for the plant hormone abscisic acid (ABA) have emerged as key targets for this application, because ABA controls the apertures of stomata, which in turn regulate transpiration. Here, we describe the rational design of cyanabactin, an ABA receptor agonist that preferentially activates <i>Pyrabactin Resistance 1</i> (PYR1) with low nanomolar potency. A 1.63 Å X-ray crystallographic structure of cyanabactin in complex with PYR1 illustrates that cyanabactin’s arylnitrile mimics ABA’s cyclohexenone oxygen and engages the tryptophan lock, a key component required to stabilize activated receptors. Further, its sulfonamide and 4-methylbenzyl substructures mimic ABA’s carboxylate and C6 methyl groups, respectively. Isothermal titration calorimetry measurements show that cyanabactin’s compact structure provides ready access to high ligand efficiency on a relatively simple scaffold. Cyanabactin treatments reduce <i>Arabidopsis</i> whole-plant stomatal conductance and activate multiple ABA responses, demonstrating that its <i>in vitro</i> potency translates to ABA-like activity <i>in vivo</i>. Genetic analyses show that the effects of cyanabactin, and the previously identified agonist quinabactin, can be abolished by the genetic removal of PYR1 and PYL1, which form subclade A within the dimeric subfamily III receptors. Thus, cyanabactin is a potent and selective agonist with a wide spectrum of ABA-like activities that defines subfamily IIIA receptors as key target sites for manipulating transpiration