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Lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG, also called 2-oxoglutarate, or 2OG)-dependent oxygenase, is an endoplasmic reticulum-resident enzyme that hydroxylates telopeptidyl lysine residues on fibrillar collagen molecules. It leads to the formation of hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which are more stable than lysine aldehyde-derived collagen cross-links (LCCs) generated devoid of LH2. It has been reported that LH2 enhances lung cancer metastatic and invasive proclivity and modulates the types of collagen cross-links (HLCC-to-LCC) in the tumor stroma. Herein, we prepared a series of 1,3-diketone analogs 1–18 and identified 12 and 13 that inhibit the LH2-driven hydroxylation of a collagen peptide substrate with IC50 approximately 300 nM and 500 nM, respectively. 12 and 13 demonstrate a 9-fold selectivity for LH2 over LH1 and LH3. Quantum Mechanics/Molecular Mechanics (QM/MM) modeling indicates that in addition to the relatively stronger interactions between compounds 12 and 13 with the active site, the selectivity stems from non-covalent interactions like hydrogen bonding between the morpholine/piperazine rings with LH2-specific Arg661, where the corresponding residue in LH1 and LH3 is Pro. Migration assays in the 344SQ lung adenocarcinoma cell line reveal that 13 shows anti-migration activity.

The catalytic function of Lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to hydroxylysine in collagen, resulting in stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs). Reports show that high amounts of LH2 lead to the accumulation of HLCCs, causing fibrosis and specific types of cancer metastasis. Some members of the Fe(II)/αKG-dependent family have also been reported to have intramolecular O2 tunnels, which aid in transporting one of the required co-substrates into the active site. While LH2 can be a promising target to combat these diseases, efficacious inhibitors are still lacking. We have used computational simulations to investigate a series of forty-four small molecules as lead compounds for LH2 inhibition. Tunneling analyses indicate the existence of several intra-molecular tunnels. The lengths of the calculated O2-transporting tunnels in holoenzymes are relatively longer than the apoenzyme suggesting that the ligands may affect the enzyme's structure and possibly block (at least partially) the tunnels. The sequence alignment analysis between LH enzymes from different organisms shows that all the amino acid residues with the highest occurrence rate in the oxygen tunnels are conserved. Our results suggest that the enolate form of diketone compounds establishes stronger interactions with the Fe(II) in the active site. Branching the enolate compounds with functional groups such as phenyl and pyridinyl enhances the interaction with various residues around the active site. Our results provide information about possible leads for further LH2 inhibition design and development.

Epithelial-to-mesenchymal transition (EMT) is a transcriptionally governed process by which cancer cells establish a front-rear polarity axis that facilitates motility and invasion. Dynamic assembly of focal adhesions and other actin-based cytoskeletal structures on the leading edge of motile cells requires precise spatial and temporal control of protein trafficking. Yet, the way in which EMT-activating transcriptional programs interface with vesicular trafficking networks that effect cell polarity change remains unclear. Here, by utilizing multiple approaches to assess vesicular transport dynamics through endocytic recycling and retrograde trafficking pathways in lung adenocarcinoma cells at distinct positions on the EMT spectrum, we find that the EMT-activating transcription factor ZEB1 accelerates endocytosis and intracellular trafficking of plasma membrane-bound proteins. ZEB1 drives turnover of the MET receptor tyrosine kinase by hastening receptor endocytosis and transport to the lysosomal compartment for degradation. ZEB1 relieves a plus-end-directed microtubule-dependent kinesin motor protein (KIF13A) and a clathrin-associated adaptor protein complex subunit (AP1S2) from microRNA-dependent silencing, thereby accelerating cargo transport through the endocytic recycling and retrograde vesicular pathways, respectively. Depletion of KIF13A or AP1S2 mitigates ZEB1-dependent focal adhesion dynamics, front-rear axis polarization, and cancer cell motility. Thus, ZEB1-dependent transcriptional networks govern vesicular trafficking dynamics to effect cell polarity change., The way in which metastatic tumour cells control endocytic vesicular trafficking networks to establish a front-rear polarity axis that facilitates motility remains unclear. Here, the authors show that the EMT activator ZEB1 influences vesicular trafficking dynamics to execute cell polarity change.

Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen’s amino- and carboxy-terminal telopeptides to create stable collagen cross-links. Here, we show that electrostatic interactions between the LH domain active site and collagen determine the unique telopeptidyl lysyl hydroxylase (tLH) activity of LH2. However, CRISPR/Cas-9-mediated inactivation of tLH activity does not fully recapitulate the inhibitory effect of LH2 knock out on LUAD growth and metastasis in mice, suggesting that LH2 drives LUAD progression, in part, through a tLH-independent mechanism. Protein homology modeling and biochemical studies identify an LH2 isoform (LH2b) that has previously undetected collagen galactosylhydroxylysyl glucosyltransferase (GGT) activity determined by a loop that enhances UDP-glucose-binding in the GLT active site and is encoded by alternatively spliced exon 13 A. CRISPR/Cas-9-mediated deletion of exon 13 A sharply reduces the growth and metastasis of LH2b-expressing LUADs in mice. These findings identify a previously unrecognized collagen GGT activity that drives LUAD progression., Guo et al. determine the molecular basis of collagen lysyl hydroxylase 2 (LH2) substrate specificity. They further show that LH2 also functions as a collagen glucosyltransferase to promote lung cancer progression.

Therapeutic strategies designed to target TP53-deficient cancer cells remain elusive. Here, we showed that TP53 loss initiated a pharmacologically actionable secretory process that drove lung adenocarcinoma (LUAD) progression. Molecular, biochemical, and cell biological studies showed that TP53 loss increased the expression of Golgi reassembly and stacking protein 55 kDa (G55), a Golgi stacking protein that maintains Golgi organelle integrity and is part of a GOLGIN45 (G45)–myosin IIA–containing protein complex that activates secretory vesicle biogenesis in the Golgi. TP53 loss activated G55-dependent secretion by relieving G55 and myosin IIA from miR-34a–dependent silencing. G55-dependent secreted proteins enhanced the proliferative and invasive activities of TP53-deficient LUAD cells and promoted angiogenesis and CD8(+) T cell exhaustion in the tumor microenvironment. A small molecule that blocks G55-G45 interactions impaired secretion and reduced TP53-deficient LUAD growth and metastasis. These results identified a targetable secretory vulnerability in TP53-deficient LUAD cells.

Heightened secretion of pro-tumorigenic effector proteins is a feature of malignant cells. Yet the molecular underpinnings and therapeutic implications of this feature remain unclear. Here we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIβ (PI4KIIIβ). Molecular, biochemical, and cell-biological studies show that PI4KIIIβ-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIβ-dependent secreted factors maintain 1q-amplified cancer cell survival and influence pro-metastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIβ antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIβ-dependent secretion for cancer cell survival and tumor progression.

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIβ (PI4KIIIβ). Molecular, biochemical, and cell biological studies show that PI4KIIIβ-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIβ-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIβ antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIβ-dependent secretion for cancer cell survival and tumor progression.

Cancer cells function as primary architects of the tumor microenvironment. Yet, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblasts (CAFs) are distinguishable on the basis of gene expression signatures they acquire in co-culture with epithelial- or mesenchymal-like lung adenocarcinoma (LUAD) cells. High expression of the EMT activator ZEB1 endows LUAD cells with the capacity to activate a soluble factor exchange that leads to CAF reprogramming, to generate CAF-led invasive projections in multicellular aggregates, and to respond to pro-metastatic signals from CAFs in mice. Thus, ZEB1-expressing LUAD cells are positioned at the apex of a signaling hierarchy in the tumor microenvironment.

SUMMARY Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis., In brief Bota-Rabassedas et al. show that EMT in lung adenocarcinoma cells activates a secretory process that governs CAF heterogeneity and, in turn, sensitizes lung adenocarcinoma cells to pro-metastatic signals from CAFs. Thus, EMT positions lung adenocarcinoma cells at the apex of a signaling hierarchy in the tumor microenvironment., Graphical Abstract

Tumor cells gain metastatic capacity through a Golgi phosphoprotein 3–dependent (GOLPH3-dependent) Golgi membrane dispersal process that drives the budding and transport of secretory vesicles. Whether Golgi dispersal underlies the pro-metastatic vesicular trafficking that is associated with epithelial-to-mesenchymal transition (EMT) remains unclear. Here, we have shown that, rather than causing Golgi dispersal, EMT led to the formation of compact Golgi organelles with improved ribbon linking and cisternal stacking. Ectopic expression of the EMT-activating transcription factor ZEB1 stimulated Golgi compaction and relieved microRNA-mediated repression of the Golgi scaffolding protein PAQR11. Depletion of PAQR11 dispersed Golgi organelles and impaired anterograde vesicle transport to the plasma membrane as well as retrograde vesicle tethering to the Golgi. The N-terminal scaffolding domain of PAQR11 was associated with key regulators of Golgi compaction and vesicle transport in pull-down assays and was required to reconstitute Golgi compaction in PAQR11-deficient tumor cells. Finally, high PAQR11 levels were correlated with EMT and shorter survival in human cancers, and PAQR11 was found to be essential for tumor cell migration and metastasis in EMT-driven lung adenocarcinoma models. We conclude that EMT initiates a PAQR11-mediated Golgi compaction process that drives metastasis.

Collagenous stromal accumulations predict a worse clinical outcome in a variety of malignancies. Better tools are needed to elucidate the way in which collagen influences cancer cells. Here, we report a method to generate collagenous matrices that are deficient in key post-translational modifications and evaluate cancer cell behaviors on those matrices. We utilized genetic and biochemical approaches to inhibit lysine hydroxylation and glucosylation on collagen produced by MC-3T3-E1 murine osteoblasts (MC cells). Seeded onto MC cell-derived matrix surface, multicellular aggregates containing lung adenocarcinoma cells alone or in combination with cancer-associated fibroblasts dissociated with temporal and spatial patterns that were influenced by collagen modifications. These findings demonstrate the feasibility of generating defined collagen matrices that are suitable for cell culture studies., Highlights • Feasibility of culturing multicellular aggregates on matrices with defined collagen modifications. • Collagen modifications influence cancer cell behavior. • This methodology is a useful tool for cancer researchers.

Lysyl hydroxylase-2 (LH2) catalyzes the hydroxylation of telopeptidyl lysine residues on collagen, leading to the formation of stable collagen cross-links that connect collagen molecules and stabilize the extracellular matrix. High levels of LH2 have been reported in the formation and stabilization of hydroxylysine aldehyde-derived collagen cross-links (HLCCs), leading to fibrosis and cancer metastasis in certain tissues. Identification of small molecule inhibitors targeting LH2 activity requires a robust and suitable assay system, which is currently lacking. Thus, despite being a promising target for these diseases, small molecule inhibitors for LH2 have yet to be reported. Therefore, we developed a luminescence-based strategy to monitor lysyl hydroxylase (LH) activity, and validated its ability to identify new inhibitors in a screen of approximately 65,000 compounds against LH2. Primary hits were confirmed using the same LH assay against mimiviral L230. This newly developed LH assay is robust, suitable for high-throughput screening, and able to identify potent specific inhibitors of LH2.

In the originally published version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to also include support from the National Institutes of Health grant T32GM008280 to Sarah Alvarado.

Collagen lysyl hydroxylases (LH1-3) are Fe2+- and 2-oxoglutarate (2-OG)-dependent oxygenases that maintain extracellular matrix homeostasis. High LH2 levels cause stable collagen cross-link accumulations that promote fibrosis and cancer progression. However, developing LH antagonists will require structural insights. Here, we report a 2 Å crystal structure and X-ray scattering on dimer assemblies for the LH domain of L230 in Acanthamoeba polyphaga mimivirus. Loop residues in the double-stranded β-helix core generate a tail-to-tail dimer. A stabilizing hydrophobic leucine locks into an aromatic tyrosine-pocket on the opposite subunit. An active site triad coordinates Fe2+. The two active sites flank a deep surface cleft that suggest dimerization creates a collagen-binding site. Loss of Fe2+-binding disrupts the dimer. Dimer disruption and charge reversal in the cleft increase Km and reduce LH activity. Ectopic L230 expression in tumors promotes collagen cross-linking and metastasis. These insights suggest inhibitor targets for fibrosis and cancer., Collagen lysyl hydroxylases promote cancer progression. Here the authors present the crystal structure of the lysyl hydroxylase domain of L230 from Acanthamoeba polyphaga mimivirus, which is of interest for LH inhibitor development, and show that ectopic expression of L230 in tumors promotes collagen cross-linking and metastasis.

Supplemental material, Supplemental_material for Development of a High-Throughput Lysyl Hydroxylase (LH) Assay and Identification of Small-Molecule Inhibitors against LH2 by Ashwini K. Devkota, John R. Veloria, Hou-Fu Guo, Jonathan M. Kurie, Eun Jeong Cho and Kevin N. Dalby in SLAS Discovery

Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, including insulin, amylin, and the amyloid β peptides. Polyanions activate IDE toward some substrates, yet an endogenous polyanion activator has not yet been identified. Here we report that inositol phosphates (InsPs) and phosphatdidylinositol phosphates (PtdInsPs) serve as activators of IDE. InsPs and PtdInsPs interact with the polyanion-binding site located on an inner chamber wall of the enzyme. InsPs activate IDE by up to ∼95-fold, affecting primarily Vmax. The extent of activation and binding affinity correlate with the number of phosphate groups on the inositol ring, with phosphate positional effects observed. IDE binds PtdInsPs from solution, immobilized on membranes, or presented in liposomes. Interaction with PtdInsPs, likely PtdIns(3)P, plays a role in localizing IDE to endosomes, where the enzyme reportedly encounters physiological substrates. Thus, InsPs and PtdInsPs can serve as endogenous modulators of IDE activity, as well as regulators of its intracellular spatial distribution.

Calcineurin is an essential serine/threonine phosphatase that plays vital roles in neuronal development and function, heart growth, and immune system activation. Calcineurin is unique in that it is the only phosphatase known to be activated by calmodulin in response to increasing intracellular calcium concentrations. Calcium-loaded calmodulin binds to the regulatory domain of calcineurin, resulting in a conformational change that removes an autoinhibitory domain from the active site of the phosphatase. We have determined a 1.95 Å crystal structure of calmodulin bound to a peptide corresponding to its binding region from calcineurin. In contrast to previous structures of this complex, our structure has a stoichiometry of 1:1 and has the canonical collapsed, wraparound conformation observed for many calmodulin-substrate complexes. In addition, we have used size-exclusion chromatography and time-resolved fluorescence to probe the stoichiometry of binding of calmodulin to a construct corresponding to almost the entire regulatory domain from calcineurin, again finding a 1:1 complex. Taken in sum, our data strongly suggest that a single calmodulin protein is necessary and sufficient to bind to and activate each calcineurin enzyme.

Bruck Syndrome is a connective tissue disease associated with inactivating mutations in lysyl hydroxylase 2 (LH2/PLOD2) or FK506 binding protein 65 (FKBP65/FKBP10). However, the functional relationship between LH2 and FKBP65 remains unclear. Here, we postulated that peptidyl prolyl isomerase (PPIase) activity of FKBP65 positively modulates LH2 enzymatic activity and is critical for the formation of hydroxylysine-aldehyde derived intermolecular collagen cross-links (HLCCs). To test this hypothesis, we analyzed collagen cross-links in Fkbp10-null and –wild-type murine embryonic fibroblasts. Although LH2 protein levels did not change, FKBP65 deficiency significantly diminished HLCCs and increased the non-hydroxylated lysine-aldehyde–derived collagen cross-links (LCCs), a pattern consistent with loss of LH2 enzymatic activity. The HLCC-to-LCC ratio was rescued in FKBP65-deficient murine embryonic fibroblasts by reconstitution with wild-type but not mutant FKBP65 that lacks intact PPIase domains. Findings from co-immunoprecipitation, protein-fragment complementation, and co-immunofluorescence assays showed that LH2 and FKBP65 are part of a common protein complex. We conclude that FKBP65 regulates LH2-mediated collagen cross-linking. Because LH2 promotes fibrosis and cancer metastasis, our findings suggest that pharmacologic strategies to target FKBP65 and LH2 may have complementary therapeutic activities.

Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens, which leads to the formation of stable collagen cross-links. Recently we reported that LH2 enhances the metastatic propensity of lung cancer by increasing the amount of stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which generate a stiffer tumor stroma (Chen, Y., et al. (2015) J. Clin. Invest. 125, 125, 1147-1162). It is generally accepted that LH2 modifies procollagen α chains on the endoplasmic reticulum before the formation of triple helical procollagen molecules. Herein, we report that LH2 is also secreted and modifies collagen in the extracellular space. Analyses of lung cancer cell lines demonstrated that LH2 is present in the cell lysates and the conditioned media in a dimeric, active form in both compartments. LH2 co-localized with collagen fibrils in the extracellular space in human lung cancer specimens and in orthotopic lung tumors generated by injection of a LH2-expressing human lung cancer cell line into nude mice. LH2 depletion in MC3T3 osteoblastic cells impaired the formation of HLCCs, resulting in an increase in the unmodified lysine aldehyde-derived collagen cross-link (LCC), and the addition of recombinant LH2 to the media of LH2-deficient MC3T3 cells was sufficient to rescue HLCC formation in the extracellular matrix. The finding that LH2 modifies collagen in the extracellular space challenges the current view that LH2 functions solely on the endoplasmic reticulum and could also have important implications for cancer biology.

The neuropilin (Nrp) family consists of essential multifunctional vertebrate cell surface receptors. Nrps were initially characterized as receptors for class III Semaphorin (Sema3) family members, functioning in axon guidance. Nrps have also been shown to be critical for vascular endothelial growth factor-dependent angiogenesis. Intriguingly, recent data show that Nrp function in these seemingly divergent pathways is critically determined by ligand-mediated cross-talk, which underlies Nrp function in both physiological and pathological processes. In addition to functioning in these two pathways, Nrps have been shown to specifically function in a number of other fundamental signaling pathways as well. Multiple general mechanisms have been found to directly contribute to the pleiotropic function of Nrp. Here we review critical general features of Nrps that function as essential receptors integrating multiple molecular cues into diverse cellular signaling.

Phagolysis of apoptotic eosinophils plays an important role in the successful resolution of asthmatic inflammation. However, little is known about underlying mechanisms. Our aim is to investigate whether annexin I is involved in the dexamethasone-mediated enhancement of phagolysis of apoptotic eosinophils by A549 cells. Phagocytosis of apoptotic eosinophils by A549 cells was visualized under laser confocal scanning microscopy. The effect of dexamethasone and TNF-alpha treatment on surface annexin I expression on A549 cells was assayed by Western blot. Eosinophils were purified under sterile conditions from periphery blood of five normal donors. A549 cells were visually assessed for apoptotic eosinophil phagocytosis by microscope. The concentration of interleukin 6 (IL-6), IL-8 and TGF-beta(1) released by A549 cells to the culture supernatants was measured by RIA or ELISA. Dexamethasone upregulated apoptotic eosinophils phagocytosis by A549 cells in a time-dependent manner, which correlated with annexin I surface expression. Annexin I mAb abolished dexamethasone-mediated enhancement of apoptotic eosinophil phagocytosis by A549 cells. Phagocytosis of apoptotic eosinophils did not change IL-6, IL-8 and TGF-beta(1) release from A549 cells. These results suggest that annexin I is involved in upregulating of dexamethasone-mediated phagocytosis of apoptotic eosinophils by A549 cells. Furthermore, the phagocytic clearance of apoptotic eosinophils did not increase proinflammatory responses.

Alzheimer's disease (AD) is the most prevalent cause of dementia in human beings. Its best-known pathologic feature is the presence of senile plaques and neurofibrillary tangles in the brain. Nogo-66 receptor (NgR) is believed to contribute to the inhibitory activities of axon regeneration after injury. This study investigated the expression of NgR in the hippocampus and its relation to the pathologic changes of AD using immunohistochemistry and double-labeling immunofluorescence methods. The results showed that NgR immunoreactivity was present in more than 50% of the pyramidal layer cells of the CA1 to CA4 subfields of the hippocampus. No significant difference was observed in the number of NgR immunopositive cells in the CA1 to CA4 subfields between patients with AD and control subjects, whereas the ratio of NgR immunopositive cells to the total number of pyramidal layer cells was revealed to be significantly higher in the CA1 and CA2 subfields of the hippocampus of patients with AD than that in the same region of the control subjects. Moreover, high numbers of AT-8 immunopositive cells were found to be double-labeled with NgR in the CA1 subfields of patients with AD, whereas only few NgR deposits were observed in the senile plaques of the hippocampus in these patients. These results suggest that NgR may be related to the formation of tangles in AD.

The Neuropilins (Nrps) are a family of essential cell surface receptors involved in multiple fundamental cellular signaling cascades. Nrp family members have key functions in VEGF-dependent angiogenesis and semaphorin-dependent axon guidance, controlling signaling and cross-talk between these fundamental physiological processes. More recently, Nrp function has been found in diverse signaling and adhesive functions, emphasizing their role as pleiotropic co-receptors. Pathological Nrp function has been shown to be important in aberrant activation of both canonical and alternative pathways. Here we review key recent insights into Nrp function in human health and disease.

Plants use the insoluble polyglucan starch as their primary glucose storage molecule. Reversible phosphorylation, at the C6 and C3 positions of glucose moieties, is the only known natural modification of starch and is the key regulatory mechanism controlling its diurnal breakdown in plant leaves. The glucan phosphatase Starch Excess4 (SEX4) is a position-specific starch phosphatase that is essential for reversible starch phosphorylation; its absence leads to a dramatic accumulation of starch in Arabidopsis, but the basis for its function is unknown. Here we describe the crystal structure of SEX4 bound to maltoheptaose and phosphate to a resolution of 1.65 A. SEX4 binds maltoheptaose via a continuous binding pocket and active site that spans both the carbohydrate-binding module (CBM) and the dual-specificity phosphatase (DSP) domain. This extended interface is composed of aromatic and hydrophilic residues that form a specific glucan-interacting platform. SEX4 contains a uniquely adapted DSP active site that accommodates a glucan polymer and is responsible for positioning maltoheptaose in a C6-specific orientation. We identified two DSP domain residues that are responsible for SEX4 site-specific activity and, using these insights, we engineered a SEX4 double mutant that completely reversed specificity from the C6 to the C3 position. Our data demonstrate that the two domains act in consort, with the CBM primarily responsible for engaging glucan chains, whereas the DSP integrates them in the catalytic site for position-specific dephosphorylation. These data provide important insights into the structural basis of glucan phosphatase site-specific activity and open new avenues for their biotechnological utilization.

Neuropilin-1 (Nrp1), an essential type I transmembrane receptor, binds two secreted ligand families, vascular endothelial growth factor (VEGF) and class III Semaphorin (Sema3). VEGF-A and Sema3F have opposing roles in regulating Nrp1 vascular function in angiogenesis. VEGF-A functions as one of the most potent pro-angiogenic cytokines, while Sema3F is a uniquely potent endogenous angiogenesis inhibitor. Sema3 family members require proteolytic processing by furin to allow competitive binding to Nrp1. We demonstrate that the furin-processed C-terminal domain of Sema3F (C-furSema) potently inhibits VEGF-A-dependent activation of endothelial cells. We find that this potent activity is due to unique heterobivalent engagement of Nrp1 by two distinct sites in the C-terminal domain of Sema3F. One of the sites is the C-terminal arginine, liberated by furin cleavage, and the other is a novel upstream helical motif centered on the intermolecular disulfide. Using a novel chimeric C-furSema, we demonstrate that combining a single C-terminal arginine with the helical motif is necessary and sufficient for potent inhibition of binding of VEGF-A to Nrp1. We further demonstrate that the multiple furin-processed variants of Sema3A, with the altered proximity of the two binding motifs, have dramatically different potencies. This suggests that furin processing not only switches Sema3 to an activated form but also, depending on the site processed, can also tune potency. These data establish the basis for potent competitive binding of Sema3 to Nrp1 and provide a basis for the design of bivalent Nrp inhibitors.

Starch is a water-insoluble, Glc-based biopolymer that is used for energy storage and is synthesized and degraded in a diurnal manner in plant leaves. Reversible phosphorylation is the only known natural starch modification and is required for starch degradation in planta. Critical to starch energy release is the activity of glucan phosphatases; however, the structural basis of dephosphorylation by glucan phosphatases is unknown. Here, we describe the structure of the Arabidopsis thaliana starch glucan phosphatase LIKE SEX FOUR2 (LSF2) both with and without phospho-glucan product bound at 2.3Å and 1.65Å, respectively. LSF2 binds maltohexaose-phosphate using an aromatic channel within an extended phosphatase active site and positions maltohexaose in a C3-specific orientation, which we show is critical for the specific glucan phosphatase activity of LSF2 toward native Arabidopsis starch. However, unlike other starch binding enzymes, LSF2 does not possess a carbohydrate binding module domain. Instead we identify two additional glucan binding sites located within the core LSF2 phosphatase domain. This structure is the first of a glucan-bound glucan phosphatase and provides new insights into the molecular basis of this agriculturally and industrially relevant enzyme family as well as the unique mechanism of LSF2 catalysis, substrate specificity, and interaction with starch granules.

Neuropilin (Nrp) receptors function as essential cell surface receptors for the Vascular Endothelial Growth Factor (VEGF) family of proangiogenic cytokines and the semaphorin 3 (Sema3) family of axon guidance molecules. There are two Nrp homologues, Nrp1 and Nrp2, which bind to both overlapping and distinct members of the VEGF and Sema3 family of molecules. Nrp1 specifically binds the VEGF-A(164/5) isoform, which is essential for developmental angiogenesis. We demonstrate that VEGF-A specific binding is governed by Nrp1 residues in the b1 coagulation factor domain surrounding the invariant Nrp C-terminal arginine binding pocket. Further, we show that Sema3F does not display the Nrp-specific binding to the b1 domain seen with VEGF-A. Engineered soluble Nrp receptor fragments that selectively sequester ligands from the active signaling complex are an attractive modality for selectively blocking the angiogenic and chemorepulsive functions of Nrp ligands. Utilizing the information on Nrp ligand binding specificity, we demonstrate Nrp constructs that specifically sequester Sema3 in the presence of VEGF-A. This establishes that unique mechanisms are used by Nrp receptors to mediate specific ligand binding and that these differences can be exploited to engineer soluble Nrp receptors with specificity for Sema3.

Living organisms utilize carbohydrates as essential energy storage molecules. Starch is the predominant carbohydrate storage molecule in plants while glycogen is utilized in animals. Starch is a water-insoluble polymer that requires the concerted activity of kinases and phosphatases to solubilize the outer surface of the glucan and mediate starch catabolism. All known plant genomes encode the glucan phosphatase Starch Excess4 (SEX4). SEX4 can dephosphorylate both the starch granule surface and soluble phosphoglucans and is necessary for processive starch metabolism. The physical basis for the function of SEX4 as a glucan phosphatase is currently unclear. Herein, we report the crystal structure of SEX4, containing phosphatase, carbohydrate-binding, and C-terminal domains. The three domains of SEX4 fold into a compact structure with extensive interdomain interactions. The C-terminal domain of SEX4 integrally folds into the core of the phosphatase domain and is essential for its stability. The phosphatase and carbohydrate-binding domains directly interact and position the phosphatase active site toward the carbohydrate-binding site in a single continuous pocket. Mutagenesis of the phosphatase domain residue F167, which forms the base of this pocket and bridges the two domains, selectively affects the ability of SEX4 to function as a glucan phosphatase. Together, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase.