An orthogonalized PYR1-based CID module with reprogrammable ligand-binding specificity.

Plants sense abscisic acid (ABA) using chemical-induced dimerization (CID) modules, including the receptor PYR1 and HAB1, a phosphatase inhibited by ligand-activated PYR1. This system is unique because of the relative ease with which ligand recognition can be reprogrammed. To expand the PYR1 system, we designed an orthogonal '*' module, which harbors a dimer interface salt bridge; X-ray crystallographic, biochemical and in vivo analyses confirm its orthogonality. We used this module to create PYR1*MANDI/HAB1* and PYR1*AZIN/HAB1*, which possess nanomolar sensitivities to their activating ligands mandipropamid and azinphos-ethyl. Experiments in Arabidopsis thaliana and Saccharomyces cerevisiae demonstrate the sensitive detection of banned organophosphate contaminants using living biosensors and the construction of multi-input/output genetic circuits. Our new modules enable ligand-programmable multi-channel CID systems for plant and eukaryotic synthetic biology that can empower new plant-based and microbe-based sensing modalities.

De novo design of small beta barrel proteins.

Small beta barrel proteins are attractive targets for computational design because of their considerable functional diversity despite their very small size (<70 amino acids). However, there are considerable challenges to designing such structures, and there has been little success thus far. Because of the small size, the hydrophobic core stabilizing the fold is necessarily very small, and the conformational strain of barrel closure can oppose folding; also intermolecular aggregation through free beta strand edges can compete with proper monomer folding. Here, we explore the de novo design of small beta barrel topologies using both Rosetta energy-based methods and deep learning approaches to design four small beta barrel folds: Src homology 3 (SH3) and oligonucleotide/oligosaccharide-binding (OB) topologies found in nature and five and six up-and-down-stranded barrels rarely if ever seen in nature. Both approaches yielded successful designs with high thermal stability and experimentally determined structures with less than 2.4 Å rmsd from the designed models. Using deep learning for backbone generation and Rosetta for sequence design yielded higher design success rates and increased structural diversity than Rosetta alone. The ability to design a large and structurally diverse set of small beta barrel proteins greatly increases the protein shape space available for designing binders to protein targets of interest.

The non-ELR CXC chemokine encoded by human cytomegalovirus UL146 genotype 5 contains a C-terminal ß-hairpin and induces neutrophil migration as a selective CXCR2 agonist.

Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. The UL146 gene exists as 14 diverse genotypes among clinical isolates, which encode 14 different CXC chemokines. One genotype (vCXCL1GT1) is a known agonist for CXCR1 and CXCR2, while two others (vCXCL1GT5 and vCXCL1GT6) lack the ELR motif considered crucial for CXCR1 and CXCR2 binding, thus suggesting another receptor targeting profile. To determine the receptor target for vCXCL1GT5, the chemokine was probed in a G protein signaling assay on all 18 classical human chemokine receptors, where CXCR2 was the only receptor being activated. In addition, vCXCL1GT5 recruited ß-arrestin in a BRET-based assay and induced migration in a chemotaxis assay through CXCR2, but not CXCR1. In contrast, vCXCL1GT1 stimulated G protein signaling, recruited ß-arrestin and induced migration through both CXCR1 and CXCR2. Both vCXCL1GT1 and vCXCL1GT5 induced equally potent and efficacious migration of neutrophils, and ELR vCXCL1GT4 and non-ELR vCXCL1GT6 activated only CXCR2. In contrast to most human chemokines, the 14 UL146 genotypes have remarkably long C-termini. Comparative modeling using Rosetta showed that each genotype could adopt the classic chemokine core structure, and predicted that the extended C-terminal tail of several genotypes (including vCXCL1GT1, vCXCL1GT4, vCXCL1GT5, and vCXCL1GT6) forms a novel ß-hairpin not found in human chemokines. Secondary NMR shift and TALOS+ analysis of vCXCL1GT1 supported the existence of two stable ß-strands. C-terminal deletion of vCXCL1GT1 resulted in a non-functional protein and in a shift to solvent exposure for tryptophan residues likely due to destabilization of the chemokine fold. The results demonstrate that non-ELR chemokines can activate CXCR2 and suggest that the UL146 chemokines have unique C-terminal structures that stabilize the chemokine fold. Increased knowledge of the structure and interaction partners of the chemokine variants encoded by UL146 is key to understanding why circulating HCMV strains sustain 14 stable genotypes.

Click-to-lead design of a picomolar ABA receptor antagonist with potent activity in vivo.

Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA's physiological roles in vivo. We set out to design antagonists that block receptor-PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an ∼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT-PYL10 complex (1.86 Å) reveals that ANT's peptidotriazole headgroup is positioned to sterically block receptor-PP2C interactions in the 4' tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA-ANT. Equilibrium dissociation constants for TAMRA-ANT binding to Arabidopsis receptors range from ∼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA-receptor function for dissecting and manipulating ABA signaling.

Structural studies of human fission protein FIS1 reveal a dynamic region important for GTPase DRP1 recruitment and mitochondrial fission.

Fission protein 1 (FIS1) and dynamin-related protein 1 (DRP1) were initially described as being evolutionarily conserved for mitochondrial fission, yet in humans the role of FIS1 in this process is unclear and disputed by many. In budding yeast where Fis1p helps to recruit the DRP1 ortholog from the cytoplasm to mitochondria for fission, an N-terminal "arm" of Fis1p is required for function. The yeast Fis1p arm interacts intramolecularly with a conserved tetratricopeptide repeat core and governs in vitro interactions with yeast DRP1. In human FIS1, NMR and X-ray structures show different arm conformations, but its importance for human DRP1 recruitment is unknown. Here, we use molecular dynamics simulations and comparisons to experimental NMR chemical shifts to show the human FIS1 arm can adopt an intramolecular conformation akin to that observed with yeast Fis1p. This finding is further supported through intrinsic tryptophan fluorescence and NMR experiments on human FIS1 with and without the arm. Using NMR, we observed the human FIS1 arm is also sensitive to environmental changes. We reveal the importance of these findings in cellular studies where removal of the FIS1 arm reduces DRP1 recruitment and mitochondrial fission similar to the yeast system. Moreover, we determined that expression of mitophagy adapter TBC1D15 can partially rescue arm-less FIS1 in a manner reminiscent of expression of the adapter Mdv1p in yeast. These findings point to conserved features of FIS1 important for its activity in mitochondrial morphology. More generally, other tetratricopeptide repeat-containing proteins are flanked by disordered arms/tails, suggesting possible common regulatory mechanisms.

Interactions between AMOT PPxY motifs and NEDD4L WW domains function in HIV-1 release.

Like most enveloped viruses, HIV must acquire a lipid membrane as it assembles and buds through the plasma membrane of infected cells to spread infection. Several sets of host cell machinery facilitate this process, including proteins of the endosomal sorting complexes required for transport pathway, which mediates the membrane fission reaction required to complete viral budding, as well as angiomotin (AMOT) and NEDD4L, which bind one another and promote virion membrane envelopment. AMOT and NEDD4L interact through the four NEDD4L WW domains and three different AMOT Pro-Pro-x (any amino acid)-Tyr (PPxY) motifs, but these interactions are not yet well defined. Here, we report that individual AMOT PPxY and NEDD4L WW domains interact with the following general affinity hierarchies: AMOT PPxY1>PPxY2>PPxY3 and NEDD4L WW3>WW2>WW1∼WW4. The unusually high-affinity of the AMOT PPxY1-NEDD4L WW3 interaction accounts for most of the AMOT-NEDD4L binding and is critical for stimulating HIV-1 release. Comparative structural, binding, and virological analyses reveal that complementary ionic and hydrophobic contacts on both sides of the WW-PPxY core interaction account for the unusually high affinity of the AMOT PPxY1-NEDD4L WW3 interaction. Taken together, our studies reveal how the first AMOT PPxY1 motif binds the third NEDD4L WW domain to stimulate HIV-1 viral envelopment and promote infectivity.

Selective Boosting of CCR7-Acting Chemokines; Short Peptides Boost Chemokines with Short Basic Tails, Longer Peptides Boost Chemokines with Long Basic Tails.

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 regulate the lymph node homing of dendritic cells and naïve T-cells and the following induction of a motile DC-T cell priming state. Although CCL19 and CCL21 bind CCR7 with similar affinities, CCL21 is a weak agonist compared to CCL19. Using a chimeric chemokine, CCL19CCL21N-term|C-term, harboring the N-terminus and the C-terminus of CCL21 attached to the core domain of CCL19, we show that these parts of CCL21 act in a synergistic manner to lower ligand potency and determine the way CCL21 engages with CCR7. We have published that a naturally occurring basic C-terminal fragment of CCL21 (C21TP) boosts the signaling of both CCL19 and CCL21. Boosting occurs as a direct consequence of C21TP binding to the CCR7 N-terminus, which seems to free chemokines with basic C-termini from an unfavorable interaction with negatively charged posttranslational modifications in CCR7. Here, we confirm this using a CCL19-variant lacking the basic C-terminus. This variant displays a 22-fold higher potency at CCR7 compared to WT CCL19 and is highly unaffected by the presence of C21TP. WT CCL19 has a short basic C-terminus, CCL21 a longer one. Here, we propose a way to differentially boost CCL19 and CCL21 activity as short and long versions of C21TP boost CCL19 activity, whereas only a long C21TP version can boost chemokines with a full-length CCL21 C-terminus.

The chemokine X-factor: Structure-function analysis of the CXC motif at CXCR4 and ACKR3.

The human chemokine family consists of 46 protein ligands that induce chemotactic cell migration by activating a family of 23 G protein-coupled receptors. The two major chemokine subfamilies, CC and CXC, bind distinct receptor subsets. A sequence motif defining these families, the X position in the CXC motif, is not predicted to make significant contacts with the receptor, but instead links structural elements associated with binding and activation. Here, we use comparative analysis of chemokine NMR structures, structural modeling, and molecular dynamic simulations that suggested the X position reorients the chemokine N terminus. Using CXCL12 as a model CXC chemokine, deletion of the X residue (Pro-10) had little to no impact on the folded chemokine structure but diminished CXCR4 agonist activity as measured by ERK phosphorylation, chemotaxis, and Gi/o-mediated cAMP inhibition. Functional impairment was attributed to over 100-fold loss of CXCR4 binding affinity. Binding to the other CXCL12 receptor, ACKR3, was diminished nearly 500-fold. Deletion of Pro-10 had little effect on CXCL12 binding to the CXCR4 N terminus, a major component of the chemokine-GPCR interface. Replacement of the X residue with the most frequent amino acid at this position (P10Q) had an intermediate effect between WT and P10del in each assay, with ACKR3 having a higher tolerance for this mutation. This work shows that the X residue helps to position the CXCL12 N terminus for optimal docking into the orthosteric pocket of CXCR4 and suggests that the CC/CXC motif contributes directly to receptor selectivity by orienting the chemokine N terminus in a subfamily-specific direction.

Specific binding-induced modulation of the XCL1 metamorphic equilibrium.

The metamorphic protein XCL1 switches between two distinct native structures with different functions in the human immune system. This structural interconversion requires complete rearrangement of all hydrogen bonding networks, yet fold-switching occurs spontaneously and reversibly in solution. One structure occupies the canonical α-ß chemokine fold and binds XCL1's cognate G-protein coupled receptor, while the other structure occupies a dimeric, all-ß fold that binds glycosaminoglycans and has antimicrobial activity. Both of these functions are important for the biologic role of XCL1 in the immune system, and each structure is approximately equally populated under near-physiologic conditions. Recent work has begun to illuminate XCL1's role in combatting infection and cancer. However, without a way to control XCL1's dynamic structural interconversion, it is difficult to study the role of XCL1 fold-switching in human health and disease. Thus, a molecular tool that can regulate the fractional population of the two XCL1 structures is needed. Here, we find by heparin affinity chromatography and NMR that an engineered XCL1 variant called CC5 can trigger a dose-dependent shift in XCL1's metamorphic equilibrium such that the receptor binding structure is depleted, and the antimicrobial structure is more heavily populated. This shift likely occurs due to formation of XCL1-CC5 heterodimers in which both protomers occupy the ß-sheet structure. These findings lay the groundwork for future studies seeking to understand the functional role of XCL1 metamorphosis, as well as studies screening for a drug-like molecule that can therapeutically target XCL1 by tuning its metamorphic equilibrium. Moreover, the proof of concept presented here suggests that protein metamorphosis is druggable, opening numerous avenues for controlling biological function of metamorphic proteins by altering the population of their multiple native states.

Agrochemical control of plant water use using engineered abscisic acid receptors.

Rising temperatures and lessening fresh water supplies are threatening agricultural productivity and have motivated efforts to improve plant water use and drought tolerance. During water deficit, plants produce elevated levels of abscisic acid (ABA), which improves water consumption and stress tolerance by controlling guard cell aperture and other protective responses. One attractive strategy for controlling water use is to develop compounds that activate ABA receptors, but agonists approved for use have yet to be developed. In principle, an engineered ABA receptor that can be activated by an existing agrochemical could achieve this goal. Here we describe a variant of the ABA receptor PYRABACTIN RESISTANCE 1 (PYR1) that possesses nanomolar sensitivity to the agrochemical mandipropamid and demonstrate its efficacy for controlling ABA responses and drought tolerance in transgenic plants. Furthermore, crystallographic studies provide a mechanistic basis for its activity and demonstrate the relative ease with which the PYR1 ligand-binding pocket can be altered to accommodate new ligands. Thus, we have successfully repurposed an agrochemical for a new application using receptor engineering. We anticipate that this strategy will be applied to other plant receptors and represents a new avenue for crop improvement.

Structural Features of an Extended C-Terminal Tail Modulate the Function of the Chemokine CCL21.

The chemokines CCL21 and CCL19, through binding of their cognate receptor CCR7, orchestrate lymph node homing of dendritic cells and naïve T cells. CCL21 differs from CCL19 via an unstructured 32 residue C-terminal domain. Previously described roles for the CCL21 C-terminus include GAG-binding, spatial localization to lymphatic vessels, and autoinhibitory modulation of CCR7-mediated chemotaxis. While truncation of the C-terminal tail induced chemical shift changes in the folded chemokine domain, the structural basis for its influence on CCL21 function remains largely unexplored. CCL21 concentration-dependent NMR chemical shifts revealed weak, nonphysiological self-association that mimics the truncation of the C-terminal tail. We generated a series of C-terminal truncation variants to dissect the C-terminus influence on CCL21 structure and receptor activation. Using NMR spectroscopy, we found that CCL21 residues 80-90 mediate contacts with the chemokine domain. In cell-based assays for CCR7 and ACKR4 activation, we also found that residues 92-100 reduced CCL21 potency in calcium flux, cAMP inhibition, and ß-arrestin recruitment. Taken together, these structure-function studies support a model wherein intramolecular interactions with specific residues of the flexible C-terminus stabilize a less active monomer conformation of the CCL21. We speculate that the autoinhibitory intramolecular contacts between the C-terminal tail and chemokine body are disrupted by GAG binding and/or interactions with the CCR7 receptor to ensure optimal functionality.

Development and Validation of 2D Difference Intensity Analysis for Chemical Library Screening by Protein-Detected NMR Spectroscopy.

An academic chemical screening approach was developed by using 2D protein-detected NMR, and a 352-chemical fragment library was screened against three different protein targets. The approach was optimized against two protein targets with known ligands: CXCL12 and BRD4. Principal component analysis reliably identified compounds that induced nonspecific NMR crosspeak broadening but did not unambiguously identify ligands with specific affinity (hits). For improved hit detection, a novel scoring metric-difference intensity analysis (DIA)-was devised that sums all positive and negative intensities from 2D difference spectra. Applying DIA quickly discriminated potential ligands from compounds inducing nonspecific NMR crosspeak broadening and other nonspecific effects. Subsequent NMR titrations validated chemotypes important for binding to CXCL12 and BRD4. A novel target, mitochondrial fission protein Fis1, was screened, and six hits were identified by using DIA. Screening these diverse protein targets identified quinones and catechols that induced nonspecific NMR crosspeak broadening, hampering NMR analyses, but are currently not computationally identified as pan-assay interference compounds. The results established a streamlined screening workflow that can easily be scaled and adapted as part of a larger screening pipeline to identify fragment hits and assess relative binding affinities in the range of 0.3-1.6 mm. DIA could prove useful in library screening and other applications in which NMR chemical shift perturbations are measured.

The aspartate-less receiver (ALR) domains: distribution, structure and function.

Two-component signaling systems are ubiquitous in bacteria, Archaea and plants and play important roles in sensing and responding to environmental stimuli. To propagate a signaling response the typical system employs a sensory histidine kinase that phosphorylates a Receiver (REC) domain on a conserved aspartate (Asp) residue. Although it is known that some REC domains are missing this Asp residue, it remains unclear as to how many of these divergent REC domains exist, what their functional roles are and how they are regulated in the absence of the conserved Asp. Here we have compiled all deposited REC domains missing their phosphorylatable Asp residue, renamed here as the Aspartate-Less Receiver (ALR) domains. Our data show that ALRs are surprisingly common and are enriched for when attached to more rare effector outputs. Analysis of our informatics and the available ALR atomic structures, combined with structural, biochemical and genetic data of the ALR archetype RitR from Streptococcus pneumoniae presented here suggest that ALRs have reorganized their active pockets to instead take on a constitutive regulatory role or accommodate input signals other than Asp phosphorylation, while largely retaining the canonical post-phosphorylation mechanisms and dimeric interface. This work defines ALRs as an atypical REC subclass and provides insights into shared mechanisms of activation between ALR and REC domains.

CCR7 Sulfotyrosine Enhances CCL21 Binding.

Chemokines are secreted proteins that direct the migration of immune cells and are involved in numerous disease states. For example, CCL21 (CC chemokine ligand 21) and CCL19 (CC chemokine ligand 19) recruit antigen-presenting dendritic cells and naïve T-cells to the lymph nodes and are thought to play a role in lymph node metastasis of CCR7 (CC chemokine receptor 7)-expressing cancer cells. For many chemokine receptors, N-terminal posttranslational modifications, particularly the sulfation of tyrosine residues, increases the affinity for chemokine ligands and may contribute to receptor ligand bias. Chemokine sulfotyrosine (sY) binding sites are also potential targets for drug development. In light of the structural similarity between sulfotyrosine and phosphotyrosine (pY), the interactions of CCL21 with peptide fragments of CCR7 containing tyrosine, pY, or sY were compared using protein NMR (nuclear magnetic resonance) spectroscopy in this study. Various N-terminal CCR7 peptides maintain binding site specificity with Y8-, pY8-, or sY8-containing peptides binding near the α-helix, while Y17-, pY17-, and sY17-containing peptides bind near the N-loop and ß3-stand of CCL21. All modified CCR7 peptides showed enhanced binding affinity to CCL21, with sY having the largest effect.

Binding of Crumbs to the Par-6 CRIB-PDZ Module Is Regulated by Cdc42.

Par-6 is a scaffold protein that organizes other proteins into a complex required to initiate and maintain cell polarity. Cdc42-GTP binds the CRIB module of Par-6 and alters the binding affinity of the adjoining PDZ domain. Allosteric regulation of the Par-6 PDZ domain was first demonstrated using a peptide identified in a screen of typical carboxyl-terminal ligands. Crumbs, a membrane protein that localizes a conserved polarity complex, was subsequently identified as a functional partner for Par-6 that likely interacts with the PDZ domain. Here we show by nuclear magnetic resonance that Par-6 binds a Crumbs carboxyl-terminal peptide and report the crystal structure of the PDZ-peptide complex. The Crumbs peptide binds Par-6 more tightly than the previously studied carboxyl peptide ligand and interacts with the CRIB-PDZ module in a Cdc42-dependent manner. The Crumbs:Par-6 crystal structure reveals specific PDZ-peptide contacts that contribute to its higher affinity and Cdc42-enhanced binding. Comparisons with existing structures suggest that multiple C-terminal Par-6 ligands respond to a common conformational switch that transmits the allosteric effects of GTPase binding.

Examination of Glycosaminoglycan Binding Sites on the XCL1 Dimer.

Known for its distinct metamorphic behavior, XCL1 interconverts between a canonical chemokine folded monomer (XCL1mon) that interacts with the receptor, XCR1, and a unique dimer (XCL1dim) that interacts with glycosaminoglycans and inhibits HIV-1 activity. This study presents the first detailed analysis of the GAG binding properties of XCL1dim. Basic residues within a conformationally selective dimeric variant of XCL1 (W55D) were mutated and analyzed for their effects on heparin binding. Mutation of Arg23 and Arg43 greatly diminished the level of heparin binding in both heparin Sepharose chromatography and surface plasmon resonance assays. To assess the contributions of different GAG structures to XCL1 binding, we developed a solution fluorescence polarization assay and correlated affinity with the length and level of sulfation of heparan sulfate oligosaccharides. It was recently demonstrated that the XCL1 GAG binding form, XCL1dim, is responsible for preventing HIV-1 infection through interactions with gp120. This study defines a GAG binding surface on XCL1dim that includes residues that are important for HIV-1 inhibition.

Structure-function analysis of CCL28 in the development of post-viral asthma.

CCL28 is a human chemokine constitutively expressed by epithelial cells in diverse mucosal tissues and is known to attract a variety of immune cell types including T-cell subsets and eosinophils. Elevated levels of CCL28 have been found in the airways of individuals with asthma, and previous studies have indicated that CCL28 plays a vital role in the acute development of post-viral asthma. Our study builds on this, demonstrating that CCL28 is also important in the chronic post-viral asthma phenotype. In the absence of a viral infection, we also demonstrate that CCL28 is both necessary and sufficient for induction of asthma pathology. Additionally, we present the first effort aimed at elucidating the structural features of CCL28. Chemokines are defined by a conserved tertiary structure composed of a three-stranded ß-sheet and a C-terminal α-helix constrained by two disulfide bonds. In addition to the four disulfide bond-forming cysteine residues that define the traditional chemokine fold, CCL28 possesses two additional cysteine residues that form a third disulfide bond. If all disulfide bonds are disrupted, recombinant human CCL28 is no longer able to drive mouse CD4+ T-cell chemotaxis or in vivo airway hyper-reactivity, indicating that the conserved chemokine fold is necessary for its biologic activity. Due to the intimate relationship between CCL28 and asthma pathology, it is clear that CCL28 presents a novel target for the development of alternative asthma therapeutics.

Activation of dimeric ABA receptors elicits guard cell closure, ABA-regulated gene expression, and drought tolerance.

Abscisic acid (ABA) is an essential molecule in plant abiotic stress responses. It binds to soluble pyrabactin resistance1/PYR1-like/regulatory component of ABA receptor receptors and stabilizes them in a conformation that inhibits clade A type II C protein phosphatases; this leads to downstream SnRK2 kinase activation and numerous cellular outputs. We previously described the synthetic naphthalene sulfonamide ABA agonist pyrabactin, which activates seed ABA responses but fails to trigger substantial responses in vegetative tissues in Arabidopsis thaliana. Here we describe quinabactin, a sulfonamide ABA agonist that preferentially activates dimeric ABA receptors and possesses ABA-like potency in vivo. In Arabidopsis, the transcriptional responses induced by quinabactin are highly correlated with those induced by ABA treatments. Quinabactin treatments elicit guard cell closure, suppress water loss, and promote drought tolerance in adult Arabidopsis and soybean plants. The effects of quinabactin are sufficiently similar to those of ABA that it is able to rescue multiple phenotypes observed in the ABA-deficient mutant aba2. Genetic analyses show that quinabactin's effects in vegetative tissues are primarily mediated by dimeric ABA receptors. A PYL2-quinabactin-HAB1 X-ray crystal structure solved at 1.98-Å resolution shows that quinabactin forms a hydrogen bond with the receptor/PP2C "lock" hydrogen bond network, a structural feature absent in pyrabactin-receptor/PP2C complexes. Our results demonstrate that ABA receptors can be chemically controlled to enable plant protection against water stress and define the dimeric receptors as key targets for chemical modulation of vegetative ABA responses.

Crystal Structure and Functional Analyses of the Lectin Domain of Glucosidase II: Insights into Oligomannose Recognition.

N-Glycans are modified as part of a quality control mechanism during glycoprotein folding in the endoplasmic reticulum (ER). Glucosidase II (GII) plays a critical role by generating monoglucosylated glycans that are recognized by lectin chaperones, calnexin and calreticulin. To understand how the hydrolytic activity of GIIα is enhanced by the mannose 6-phosphate receptor (MPR) homology domain (MRH domain) of its ß subunit, we now report a 1.6 Å resolution crystal structure of the MRH domain of GIIß bound to mannose. A comparison of ligand-bound and unbound structures reveals no major difference in their overall fold, but rather a repositioning of side chains throughout the binding pocket, including Y372. Mutation of Y372 inhibits GII activity, demonstrating an important role for Y372 in regulating GII activity. Comparison of the MRH domains of GIIß, MPRs, and the ER lectin OS-9 identified conserved residues that are critical for the structural integrity and architecture of the carbohydrate binding pocket. As shown by nuclear magnetic resonance spectroscopy, mutations of the primary binding pocket residues and adjacent W409, all of which inhibit the activity of GII both in vitro and in vivo, do not cause a significant change in the overall fold of the GIIß MRH domain but impact locally the stability of the binding pocket. W409 does not directly contact mannose; rather, its indole ring is stabilized by binding into a hydrophobic pocket of an adjacent crystallographic neighbor. This suggests that W409 interacts with a hydrophobic region of the GIIß or GIIα subunit to modulate its effect on GII activity.

Solution Structure of CCL19 and Identification of Overlapping CCR7 and PSGL-1 Binding Sites.

CCL19 and CCL21 are chemokines involved in the trafficking of immune cells, particularly within the lymphatic system, through activation of CCR7. Concurrent expression of PSGL-1 and CCR7 in naive T-cells enhances recruitment of these cells to secondary lymphoid organs by CCL19 and CCL21. Here the solution structure of CCL19 is reported. It contains a canonical chemokine domain. Chemical shift mapping shows the N-termini of PSGL-1 and CCR7 have overlapping binding sites for CCL19 and binding is competitive. Implications for the mechanism of PSGL-1's enhancement of resting T-cell recruitment are discussed.