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This dataset contains the supplementary materialsfor "De novo design of small beta barrel proteins" Datasets. All dataset_SX.tar.gz files extract into a directory named data_archive. Dataset S1 (dataset_S1.tar.gz). Contains designs, input files (including blueprints, constraints, resfiles, and Rosetta xml scripts), commands, and selection scripts for Batch1-5 designs. data_archive/batch1 data_archive/batch2 data_archive/batch3 data_archive/batch4 data_archive/batch5 Dataset S2 (dataset_S2.tar.gz). Contains sequences, designs, input files (including blueprints, constraints, resfiles, and Rosetta xml scripts), commands, selection scripts, and TrRosetta and DeepAccNet (DAN) scores for chip library designs. data_archive/chip Dataset S3 (dataset_S3.tar.gz). Contains chip library protease stability assay data including sequence counts, protease EC50 and stability scores, selection results (experiments.csv), and experimental meta data. data_archive/protease_assay Dataset S4 (dataset_S4.tar.gz). Contains the LASSO regression model and feature data used for training from the chip library protease stability screen results and for predicting protease stability for the constrained hallucinated designs. data_archive/lasso_regression_model Dataset S5 (dataset_S5.tar.gz). Contains the mass spectrometry barcode library data (skyline.csv). data_archive/ms_barcodes Dataset S6 (dataset_S6.tar.gz). Contains common input files, the fragment database in HDF5 format used for structure profiles (PSSMs), command and inputs for Rosetta design filter metrics, and a script to extract DeepAccNet (DAN) scores. data_archive/common_inputs data_archive/databases/ss_grouped_vall_all.h5 data_archive/filter data_archive/deepaccnet Dataset S7 (dataset_S7.tar.gz). Contains native small beta barrel PDB sets and the clustering script used for selection. data_archive/natives Dataset S8 (dataset_S8.tar.gz). Contains a scaffold set of 12911 designed mini-barrels which have a high probability of folding based on AlphaFold2 metrics (pLDDT > 88 and model RMSD < 2.0). data_archive/scaffolds &nbsp

G protein–coupled receptors (GPCRs) recruit β-arrestins to coordinate diverse cellular processes, but the structural dynamics driving this process are poorly understood. Atypical chemokine receptors (ACKRs) are intrinsically biased GPCRs that engage β-arrestins but not G proteins, making them a model system for investigating the structural basis of β-arrestin recruitment. Here, we performed nuclear magnetic resonance (NMR) experiments on 13 CH 3 -ε–methionine–labeled ACKR3, revealing that β-arrestin recruitment is associated with conformational exchange at key regions of the extracellular ligand-binding pocket and intracellular β-arrestin–coupling region. NMR studies of ACKR3 mutants defective in β-arrestin recruitment identified an allosteric hub in the receptor core that coordinates transitions among heterogeneously populated and selected conformational states. Our data suggest that conformational selection guides β-arrestin recruitment by tuning receptor dynamics at intracellular and extracellular regions.

Recurrence of therapy-resistant tumors is a principal problem in solid tumor oncology, particularly in ovarian cancer. Despite common complete responses to first line, platinum-based therapies, most women with ovarian cancer recur, and eventually, nearly all with recurrent disease develop platinum resistance. Likewise, both intrinsic and acquired resistance contribute to the dismal prognosis of pancreatic cancer. Our previous work and that of others has established CLPTM1L (cleft lip and palate transmembrane protein 1-like)/CRR9 (cisplatin resistance related protein 9) as a cytoprotective oncofetal protein that is present on the tumor cell surface. We show that CLPTM1L is broadly overexpressed and accumulated on the plasma membrane of ovarian tumor cells, while weakly or not expressed in normal tissues. High expression of CLPTM1L is associated with poor outcome in ovarian serous adenocarcinoma. Robust re-sensitization of resistant ovarian cancer cells to platinum-based therapy was achieved using human monoclonal biologics inhibiting CLPTM1L in both orthotopic isografts and patient-derived cisplatin resistant xenograft models. Furthermore, we demonstrate that in addition to cell-autonomous cytoprotection by CLPTM1L, extracellular CLPTM1L confers resistance to chemotherapeutic killing in an ectodomain-dependent fashion, and that this intercellular resistance mechanism is inhibited by anti-CLPTM1L biologics. Specifically, exosomal CLPTM1L from cisplatin-resistant ovarian carcinoma cell lines conferred resistance to cisplatin in drug-sensitive parental cell lines. CLPTM1L is present in extracellular vesicle fractions of tumor culture supernatants and in patients’ serum with increasing abundance upon chemotherapy treatment. These findings have encouraging implications for the use of anti-CLPTM1L targeted biologics in the treatment of therapy-resistant tumors.

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.

A general method to generate biosensors for user-defined molecules could provide detection tools for a wide range of biological applications. Here, we describe an approach for the rapid engineering of biosensors using PYR1 (Pyrabactin Resistance 1), a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and a requirement for ligand-induced heterodimerization, which facilitates the construction of sense–response functions. We applied this platform to evolve 21 sensors with nanomolar to micromolar sensitivities for a range of small molecules, including structurally diverse natural and synthetic cannabinoids and several organophosphates. X-ray crystallography analysis revealed the mechanistic basis for new ligand recognition by an evolved cannabinoid receptor. We demonstrate that PYR1-derived receptors are readily ported to various ligand-responsive outputs, including enzyme-linked immunosorbent assay (ELISA)-like assays, luminescence by protein-fragment complementation and transcriptional circuits, all with picomolar to nanomolar sensitivity. PYR1 provides a scaffold for rapidly evolving new biosensors for diverse sense–response applications.

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 G(i/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.

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 β-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.

Chemoattractant gradients frequently guide migrating cells. To achieve the most directional signal, such gradients should be maintained with concentrations around the dissociation constant (Kd)1–6 of the chemoreceptor. Whether this actually occurs in animals is unknown. Here we investigate whether a moving tissue, the zebrafish posterior lateral line primordium, buffers its attractant in this concentration range to achieve robust migration. We find that the Cxcl12 (also known as Sdf1) attractant gradient ranges from 0 to 12 nM, values similar to the 3.4 nM Kd of its receptor Cxcr4. When we increase the Kd of Cxcl12 for Cxcr4, primordium migration is less directional. Furthermore, a negative-feedback loop between Cxcl12 and its clearance receptor Ackr3 (also known as Cxcr7) regulates the Cxcl12 concentrations. Breaking this negative feedback by blocking the phosphorylation of the cytoplasmic tail of Ackr3 also results in less directional primordium migration. Thus, directed migration of the primordium is dependent on a close match between the Cxcl12 concentration and the Kd of Cxcl12 for Cxcr4, which is maintained by buffering of the chemokine levels. Quantitative modelling confirms the plausibility of this mechanism. We anticipate that buffering of attractant concentration is a general mechanism for ensuring robust cell migration. Lau et al. quantify endogenous concentrations of the chemokine Cxcl12 and its binding affinity for its cognate receptor Cxcr4 in zebrafish embryos, uncovering a negative-feedback loop governing directional cell migration in vivo.

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.

Background Chemokines represent a superfamily of immune-modulatory small protein molecules that regulate leukocyte migration to inflammatory sites through their chemoattractant and cell signaling properties. This review focuses on the immunological functions of the CCR6 chemokine receptor and is chemokine ligand, CCL20, that contribute to it role in inflammation in human psoriasis. Methods Peer-reviewed relevant articles are searched and selected from 2000 to 2022 using the search engines including PubMed and Google Scholar. Results After selectively reviewing and evaluating over seventy articles, a comprehensive overview on the immunology of CCL20-CCR6 axis in psoriasis and psoriatic arthritis, the X-ray crystal structures of CCL20 monomers, and the potential of developing clinical therapies targeting this axis is summarized. Conclusions Over the past decade, preclinical studies carried out in animal models of psoriasis involving agents targeting CCL20-CCR6 axis have yielded promising results. Other studies that this axis may play a role in a number of other autoimmune diseases, including rheumatoid arthritis, suggesting a rationale for further investigation into this key signaling/migratory pathway.

Significance Abscisic acid (ABA) is a phytohormone that plants utilize to coordinate responses to abiotic stress, modulate seed dormancy, and is central to plant development in several contexts. Chemicals that activate or block ABA signaling are useful as research tools and as potential agrochemical leads. Many successes have been reported for ABA activators (agonists), but existing ABA blockers (antagonists) are limited by modest in vivo activity. Here we report antabactin (ANT), a potent ABA blocker developed using “click chemistry”–based diversification of a known ABA activator. Structural studies reveal, ANT disrupts signaling by stabilizing ABA receptors in an unproductive form. ANT can accelerate seed germination in multiple species, making it a chemical tool for improving germination., 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.

The pleiotropic chemokine CXCL12 is involved in diverse physiological and pathophysiological processes, including embryogenesis, hematopoiesis, leukocyte migration, and tumor metastasis. It is known to engage the classical receptor CXCR4 and the atypical receptor ACKR3. Differential receptor engagement can transduce distinct cellular signals and effects as well as alter the amount of free, extracellular chemokine. CXCR4 binds both monomeric and the more commonly found dimeric forms of CXCL12, whereas ACKR3 binds monomeric forms. Here, we found that CXCL12 also bound to the atypical receptor ACKR1 (previously known as Duffy antigen/receptor for chemokines or DARC). In vitro nuclear magnetic resonance spectroscopy and isothermal titration calorimetry revealed that dimeric CXCL12 bound to the extracellular N terminus of ACKR1 with low nanomolar affinity, whereas the binding affinity of monomeric CXCL12 was orders of magnitude lower. In transfected MDCK cells and primary human Duffy-positive erythrocytes, a dimeric, but not a monomeric, construct of CXCL12 efficiently bound to and internalized with ACKR1. This interaction between CXCL12 and ACKR1 provides another layer of regulation of the multiple biological functions of CXCL12. The findings also raise the possibility that ACKR1 can bind other dimeric chemokines, thus potentially further expanding the role of ACKR1 in chemokine retention and presentation. published

Mitochondrial dysfunction is one of several factors that drive development of vascular endothelial dysfunction in type 2 diabetes (T2DM). In endothelial cells from T2DM patients, mitochondrial networks are highly fragmentated with increased expression of mitochondrial fission protein 1 (Fis1). However, whether manipulation of Fis1 expression and activity in endothelial vessels from T2DM patients alters endothelial function remains unknown. Here, molecular suppression of Fis1 reversed impaired endothelium-dependent vasodilation of vessels from T2DM patients, as well as healthy human vessels exposed to high (33 mM) or low (2.5 mM) glucose, while preserving NO bioavailability and improving endothelial cell layer integrity. Conversely, overexpression of Fis1 in healthy vessels impaired vasodilation and increased mitochondrial superoxide, suggesting a causative role. Application of a novel and specific Fis1 inhibitor, pep213, improved endothelium-dependent vasodilation of vessels from T2DM patients, as well as healthy vessels exposed to high glucose or Fis1 overexpression, by improving NO bioavailability and decreasing excess mitochondrial ROS generation. The specificity of pep213 was determined through multiple biophysical techniques and a 1.85 Å crystal structure of pep213 in complex with Fis1. These data support that excessive mitochondrial fragmentation drives endothelial vessel dysfunction and supports a potential novel therapeutic route for treating diabetic microvascular disease through pharmacological inhibition of Fis1.

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-β chemokine fold and binds XCL1’s cognate G-protein coupled receptor, while the other structure occupies a dimeric, all-β 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 β-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.

One sequence encoding two structures Most proteins have stable, folded structures, but there are rare examples of metamorphic proteins that can switch between two different folds that may each have a different function. Dishman et al. investigated the evolution of XCL1, which is a member of the chemokine family that interconverts between the chemokine fold and a second, noncanonical fold that forms dimers. The authors used nuclear magnetic resonance spectroscopy to investigate the structures of inferred evolutionary ancestral sequences. Their results suggest that XCL1 evolved from an ancestor with the chemokine fold and then transitioned to prefer the noncanonical fold before reaching the modern-day metamorphic protein. Science , this issue p. 86

The chemokine CCL28 is constitutively expressed in mucosal tissues and is abundant in low-salt mucosal secretions. Beyond its traditional role as a chemoattractant, CCL28 has been shown to act as a potent and broad-spectrum antimicrobial agent with particular efficacy against the commensal fungus and opportunistic pathogen Candida albicans. However, the structural features that allow CCL28 to perform its chemotactic and antimicrobial functions remain unknown. Here, we report the structure of CCL28, solved using nuclear magnetic resonance spectroscopy. CCL28 adopts the canonical chemokine tertiary fold, but also has a disordered C-terminal domain that is partially tethered to the core by a non-conserved disulfide bond. Structure-function analysis reveals that removal of the C-terminal tail reduces the antifungal activity of CCL28 without disrupting its structural integrity. Conversely, removal of the non-conserved disulfide bond destabilizes the tertiary fold of CCL28 without altering its antifungal effects. Moreover, we report that CCL28 unfolds in response to low pH but is stabilized by the presence of salt. To explore the physiologic relevance of the observed structural lability of CCL28, we investigated the effects of pH and salt on the antifungal activity of CCL28 in vitro. We found that low pH enhances the antifungal potency of CCL28, but also that this pH effect is independent of CCL28's tertiary fold. Given its dual role as a chemoattractant and antimicrobial agent, our results suggest that changes in the salt concentration or pH at mucosal sites may fine-tune CCL28's functional repertoire by adjusting the thermostability of its structure.

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 cross-peak broadening, hampering NMR analyses, but are currently not computationally identified as pan-assay interference compounds. The results established a streamlined screening work-flow 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.

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.

Psoriasis is a chronic inflammatory skin disease characterized by the infiltration of T cell and other immune cells to the skin in response to injury or autoantigens. Conventional, as well as unconventional, γδ T cells are recruited to the dermis and epidermis by CCL20 and other chemokines. Together with its receptor CCR6, CCL20 plays a critical role in the development of psoriasiform dermatitis in mouse models. We screened a panel of CCL20 variants designed to form dimers stabilized by intermolecular disulfide bonds. A single-atom substitution yielded a CCL20 variant (CCL20 S64C) that acted as a partial agonist for the chemokine receptor CCR6. CCL20 S64C bound CCR6 and induced intracellular calcium release, consistent with G-protein activation, but exhibited minimal chemotactic activity. Instead, CCL20 S64C inhibited CCR6-mediated T cell migration with nominal impact on other chemokine receptor signaling. When given in an IL-23-dependent mouse model for psoriasis, CCL20 S64C prevented psoriatic inflammation and the up-regulation of IL-17A and IL-22. Our results validate CCR6 as a tractable therapeutic target for psoriasis and demonstrate the value of CCL20 S64C as a lead compound.

Enterococcus faecalis, a leading cause of hospital-acquired infections, exhibits intrinsic resistance to most cephalosporins, which are antibiotics in the beta-lactam family that target cell-wall biosynthesis. A comprehensive understanding of the underlying genetic and biochemical mechanisms of cephalosporin resistance in E. faecalis is lacking. We previously determined that a transmembrane serine-threonine kinase (IreK) and its cognate phosphatase (IreP) reciprocally regulate cephalosporin resistance in E. faecalis, dependent on the kinase activity of IreK. Other than IreK itself, thus far the only known substrate for reversible phosphorylation by IreK and IreP is IreB, a small protein of unknown function that is well conserved in low-GC Gram-positive bacteria. We previously showed that IreB acts as a negative regulator of cephalosporin resistance in E. faecalis. However, the biochemical mechanism by which IreB modulates cephalosporin resistance remains unknown. As a next step towards an understanding of the mechanism by which IreB regulates resistance, we initiated a structure-function study on IreB. The NMR solution structure of IreB was determined, revealing that IreB adopts a unique fold and forms a dimer in vitro. Dimerization of IreB was confirmed in vivo. Substitutions at the dimer interface impaired IreB function and stability in vivo, indicating that dimerization is functionally important for the biological activity of IreB. Hence, these studies provide new insights into the structure and function of a widely conserved protein of unknown function that is an important regulator of antimicrobial resistance in E. faecalis.

The interaction of high density lipoprotein (HDL) with its receptor, scavenger receptor BI (SR-BI), is critical for lowering plasma cholesterol levels and reducing the risk for cardiovascular disease. The HDL/SR-BI complex facilitates delivery of cholesterol into cells and is likely mediated by receptor dimerization. This work describes the use of nuclear magnetic resonance (NMR) spectroscopy to generate the first high-resolution structure of the C-terminal transmembrane domain of SR-BI. This region of SR-BI harbors a leucine zipper dimerization motif, that when mutated, impairs the ability of the receptor to bind HDL and mediate cholesterol delivery. These losses in function correlate with the inability of SR-BI to form dimers. We also identify juxtamembrane regions of the extracellular domain of SR-BI that may interact with the lipid surface to facilitate cholesterol transport functions of the receptor.

Plant thirst quenched without water Drought causes many billions of dollars of annual losses to farmers worldwide. Central to a plant's water use efficiency are signaling pathways regulated by the hormone abscisic acid and its receptors. Vaidya et al. screened a pool of candidate small molecules and used structure-guided design to optimize the function of an abscisic acid receptor agonist (see the Perspective by Phillips and Sussman). Application of the agonist protected Arabidopsis , wheat, and tomato from underwatering. Science , this issue p. eaaw8848 ; see also p. 416

The past decade has witnessed remarkable progress in the determination of G protein-coupled receptor (GPCR) structures, profoundly expanding our understanding of how GPCRs recognize ligands, become activated, and interact with intracellular signaling components. In recent years, numerous studies have used solution nuclear magnetic resonance (NMR) spectroscopy to investigate GPCRs, providing fundamental insights into GPCR conformational changes, allostery, dynamics, and other facets of GPCR function are challenging to study using other structural techniques. Despite these advantages, NMR-based studies of GPCRs are few relative to the number of published structures, due in part to the challenges and limitations of NMR for the characterization of large membrane proteins. Several studies have circumvented these challenges using a variety of isotopic labeling strategies, including side chain derivatization and metabolic incorporation of NMR-active nuclei. In this chapter, we provide an overview of different isotopic labeling strategies and describe an in-depth protocol for the expression, purification, and NMR studies of the chemokine GPCR atypical chemokine receptor 3 (ACKR3) via 13CH3-methionine incorporation. The goal of this chapter is to provide a resource to the GPCR community for those interested in pursuing NMR studies of GPCRs.

Tyrosine sulfation is a post-translational modification that facilitates protein-protein interaction. Two sulfated tyrosines (Tys173 and Tys177) were recently identified within the second variable (V2) loop of the major HIV-1 envelope glycoprotein, gp120, and shown to contribute to stabilizing the intramolecular interaction between V2 and the third variable (V3) loop. Here, we report that tyrosine-sulfated peptides derived from V2 act as structural and functional mimics of the CCR5 N-terminus and potently block HIV-1 infection. Nuclear magnetic and surface plasmon resonance analyses indicate that a tyrosine-sulfated V2 peptide (pV2α-Tys) adopts a CCR5-like helical conformation and directly interacts with gp120 in a CD4-dependent fashion, competing with a CCR5 N-terminal peptide. Sulfated V2 mimics, but not their non-sulfated counterparts, inhibit HIV-1 entry and fusion by preventing coreceptor utilization, with the highly conserved C-terminal sulfotyrosine, Tys177, playing a dominant role. Unlike CCR5 N-terminal peptides, V2 mimics inhibit a broad range of HIV-1 strains irrespective of their coreceptor tropism, highlighting the overall structural conservation of the coreceptor-binding site in gp120. These results document the use of receptor mimicry by a retrovirus to occlude a key neutralization target site and provide leads for the design of therapeutic strategies against HIV-1., Highlights • Tyrosine-sulfated peptides derived from the V2 domain of HIV-1 gp120 mimic the N-terminal domain of the CCR5 coreceptor. • Tyrosine-sulfated V2 peptides are potent and broad-spectrum inhibitors of HIV-1 infection. Understanding how HIV-1 protects its outer envelope from the immune system may help devise effective strategies for treatment and vaccine. We derived synthetic peptides from the V2 loop of the external HIV-1 envelope glycoprotein, gp120, which contains sulfate-modified tyrosines that contribute to maintaining the envelope in an antibody-protected configuration. We found that these peptides mimic the structure and function of CCR5, a key cellular coreceptor for HIV-1, interacting with and occluding a major CCR5-binding site in gp120. Tyrosine-sulfated V2 peptides potently block HIV-1 entry and may serve as templates for the design of new antiviral inhibitors.

Scavenger receptor class B type I (SR-BI), the high density lipoprotein (HDL) receptor, is important for the delivery of HDL–cholesteryl esters to the liver for excretion via bile formation. The focus on therapeutic strategies aimed at reducing cholesterol levels highlights the critical need to understand the structural features of SR-BI that drive cholesterol removal. Yet, in the absence of a high-resolution structure of SR-BI, our understanding of how SR-BI interacts with HDL is limited. In this study, we have optimized the NMR solution conditions for the structural analysis of the C-terminal transmembrane domain of SR-BI that harbors putative domains required for receptor oligomerization. An isotopically-labeled SR-BI peptide encompassing residues 405–475 was bacterially-expressed and purified. [ U - 15 N]-SR-BI(405–475) was incorporated into different detergent micelles and assessed by 1 H– 15 N-HSQC in order to determine which detergent micelle best maintained SR-BI(405–475) in a folded, native conformation for subsequent NMR analyses. We also determined the optimal detergent concentration used in micelles, as well as temperature, solution buffer and pH conditions. Based on 1 H– 15 N-HSQC peak dispersion, intensity, and uniformity, we determined that [ U - 15 N]-SR-BI(405–475) should be incorporated into 5% detergent micelles consisting of 1-palmitoyl-2-hydroxy- sn -glycero-3-phospho-[1′-rac-glycerol] (LPPG) and data collected at 40 °C in a non-buffered solution at pH 6.8. Furthermore, we demonstrate the ability of SR-BI(405–475) to form dimers upon chemical crosslinking. These studies represent the first steps in obtaining high-resolution structural information by NMR for the HDL receptor that plays a critical role in regulating whole body cholesterol removal.

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 Pyrabactin Resistance 1 (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 Arabidopsis whole-plant stomatal conductance and activate multiple ABA responses, demonstrating that its in vitro potency translates to ABA-like activity in vivo. 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.

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 β3-stand of CCL21. All modified CCR7 peptides showed enhanced binding affinity to CCL21, with sY having the largest effect.

Chemokines orchestrate cell migration for development, immune surveillance, and disease by binding to cell surface heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs). The array of interactions between the nearly 50 chemokines and their 20 GPCR targets generates an extensive signaling network to which promiscuity and biased agonism add further complexity. The receptor CXCR4 recognizes both monomeric and dimeric forms of the chemokine CXCL12, which is a distinct example of ligand bias in the chemokine family. We demonstrated that a constitutively monomeric CXCL12 variant reproduced the G protein–dependent and β-arrestin–dependent responses that are associated with normal CXCR4 signaling and lead to cell migration. In addition, monomeric CXCL12 made specific contacts with CXCR4 that are not present in the structure of the receptor in complex with a dimeric form of CXCL12, a biased agonist that stimulates only G protein–dependent signaling. We produced an experimentally validated model of an agonist-bound chemokine receptor that merged a nuclear magnetic resonance–based structure of monomeric CXCL12 bound to the amino terminus of CXCR4 with a crystal structure of the transmembrane domains of CXCR4. The large CXCL12:CXCR4 protein-protein interface revealed by this structure identified previously uncharacterized functional interactions that fall outside of the classical “two-site model” for chemokine-receptor recognition. Our model suggests a mechanistic hypothesis for how interactions on the extracellular face of the receptor may stimulate the conformational changes required for chemokine receptor-mediated signal transduction.

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.

CXCL12 binds to CXCR4, promoting both chemotaxis of lymphocytes and metastasis of cancer cells. We previously identified small molecule ligands that bind CXCL12 and block CXCR4-mediated chemotaxis. We now report a 1.9 Å resolution X-ray structure of CXCL12 bound by such a molecule at a site normally bound by sY21 of CXCR4. The complex structure reveals binding hot spots for future inhibitor design and suggests a new approach to targeting CXCL12–CXCR4 signaling in drug discovery.

Brugia malayi is a parasitic nematode that causes lymphatic filariasis in humans. Here the solution structure of the forkhead DNA binding domain of Brugia malayi DAF-16a, a putative ortholog of Caenorhabditis elegans DAF-16, is reported. It is believed to be the first structure of a forkhead or winged helix domain from an invertebrate. C. elegans DAF-16 is involved in the insulin/IGF-I signaling pathway and helps control metabolism, longevity, and development. Conservation of sequence and structure with human FOXO proteins suggests that B. malayi DAF-16a is a member of the FOXO family of forkhead proteins.

Medullary thyroid carcinoma (MTC) is a neoplasm of the endocrine system, which originates from parafollicular C-cells of the thyroid gland. For MTC therapy, the Food and Drug Administration recently approved vandetanib and cabozantinib, multi-kinase inhibitors targeting RET and other tyrosine kinase receptors of vascular endothelial growth factor, epidermal growth factor, or hepatocyte growth factor. Nevertheless, not all patients with the progressive MTC respond to these drugs, requiring the development of additional therapeutic modalities that have distinct activity. Previously, we reported that expression of activated Ras or Raf in the human MTC cell lines, TT and MZ-CRC-1, can induce growth arrest and RET downregulation via a leukemia inhibitory factor (LIF)-mediated autocrine/paracrine loop. In this study, we aimed to evaluate bacterially-produced recombinant human LIF for its efficacy to suppress human MTC xenografts in mice. Here, we report that, consistent with its effects in vitro, locally or systemically administered recombinant LIF effectively suppressed growth of TT and MZ-CRC-1 xenografts in mice. Further, as predicted from its effects in TT and MZ-CRC-1 cell cultures in vitro, recombinant LIF activated the JAK/STAT pathway and downregulated RET and E2F1 expression in tumors in mice. These results suggest that LIF is a potent cytostatic agent for MTC cells, which regulates unique mechanisms that are not targeted by currently available therapeutic agents.

Numerous Gram-negative bacterial pathogens use type III secretion systems to deliver effector molecules into the cytoplasm of a host cell. Many of these effectors have evolved to manipulate the host ubiquitin system to alter host cell physiology or the location, stability, or function of the effector itself. ExoU is a potent A2 phospholipase used by Pseudomonas aeruginosa to destroy membranes of infected cells. The enzyme is held in an inactive state inside of the bacterium due to the absence of a required eukaryotic activator, which was recently identified as ubiquitin. This study sought to identify the region of ExoU required to mediate this interaction and determine the properties of ubiquitin important for binding, ExoU activation, or both. Biochemical and biophysical approaches were used to map the ubiquitin-binding domain to a C-terminal four-helix bundle of ExoU. The hydrophobic patch of ubiquitin is required for full binding affinity and activation. Binding and activation were uncoupled by introducing an L8R substitution in ubiquitin. Purified L8R demonstrated a parental binding phenotype to ExoU but did not activate the phospholipase in vitro. Utilizing these new biochemical data and intermolecular distance measurements by double electron-electron resonance, we propose a model for an ExoU-monoubiquitin complex. Background: Efficient activation of ExoU is dependent upon its interaction with ubiquitin. Results: C-terminal regions, distal from the catalytic domain, mediate the highest affinity interaction with the hydrophobic patch of ubiquitin. Conclusion: The C-terminal domain of ExoU has multiple functions critical to activity. Significance: This is the first report characterizing the cofactor-binding domain of a bacterial toxin activated by ubiquitin.

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.

CXCL12 is a human chemokine that recognizes the CXCR4 receptor and is involved in immune responses and metastatic cancer. Interactions between CXCL12 and CXCR4 are an important drug target but, like other elongated protein-protein interfaces, present challenges for small molecule ligand discovery due to the relatively shallow and featureless binding surfaces. Calculations using an NMR complex structure revealed a binding hot spot on CXCL12 that normally interacts with the I4/I6 residues from CXCR4. Virtual screening was performed against the NMR model, and subsequent testing has verified the specific binding of multiple docking hits to this site. Together with our previous results targeting two other binding pockets that recognize sulfotyrosine residues (sY12 and sY21) of CXCR4, including a new analog against the sY12 binding site reported herein, we demonstrate that protein-protein interfaces can often possess multiple sites for engineering specific small molecule ligands that provide lead compounds for subsequent optimization by fragment based approaches.

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.

Chemokines are small, secreted proteins that bind G protein-coupled receptors (GPCRs) to orchestrate cellular chemotaxis in the immune system and beyond. While the ∼50 members of the chemokine family share a remarkably conserved tertiary structure, they still manage to coordinate diverse, non-redundant physiologic and pathologic functions in the human body. It is therefore assumed that more subtle nuances in the structure and dynamics of each chemokine-receptor pair are the key determinants of their biologic function. Here we present the molecular structure of CCL28, an especially intriguing chemokine recently identified to play a major role in asthma pathogenesis, solved by solution-state nuclear magnetic resonance spectroscopy (NMR). The structure reveals that while CCL28 adopts the conserved chemokine tertiary fold, it has a particularly curious structural feature not seen in other chemokines. An intrinsically disordered C-terminal domain of ∼30 residues extends from the globular core of the chemokine, part of which is tethered by a non-conserved disulfide bond. Dynamic NMR experiments including backbone 1H-15N heteronuclear NOE measurements, heteronuclear single quantum coherence (HSQC) titrations, and pulsed field gradient (PFG) diffusion measurements collectively indicate the disordered, dynamic nature of the C-terminal extension. Combined with mutational and in vitro functional studies, our findings suggest that the unique structural and dynamic features of CCL28 may be key determinants of its pathologic function.

The diverse roles of chemokines in normal immune function and many human diseases have motivated numerous investigations into the structure and function of this family of proteins. Recombinant chemokines are often used to study how chemokines coordinate the trafficking of immune cells in various biological contexts. A reliable source of biologically active protein is vital for any in vitro or in vivo functional analysis. In this chapter, we describe a general method for the production of recombinant chemokines and robust techniques for efficient refolding that ensure consistently high biological activity. Considerations for initiating development of protocols consistent with Current Good Manufacturing Practices (cGMPs) to produce biologically active chemokines suitable for use in clinical trials are also discussed.

The equilibrium unfolding reaction of Ltn, a metamorphic C-class chemokine, was monitored by tryptophan fluorescence to determine unfolding free energies. Measurements revealed that addition of 150 mM NaCl stabilized the Ltn chemokine fold by approximately 1 kcal/mol. Specific mutations involving Arg23 and Arg43 also increased the stability by 1 kcal/mol, suggesting their involvement in chloride ion coordination. This interaction was confirmed by nuclear magnetic resonance (NMR) salt titration studies that revealed chemical shift perturbations localized to these residues and backbone amides within the proximal 40s loop. The effects of NaCl on the free energy landscape were further verified by ZZ-exchange NMR spectroscopy. Our results suggest that changes in the electrostatic environment modulate the Gibbs free energy of folding and alter the forward and reverse rates of interconversion. These results demonstrate how solution ions can promote metamorphic folding by adjusting the relative stabilities of two unrelated Ltn native-state structures.