High-pressure EPR spectroscopy studies of the E. coli lipopolysaccharide transport proteins LptA and LptC.

The use of pressure is an advantageous approach to the study of protein structure and dynamics because it can shift the equilibrium populations of protein conformations toward higher energy states that are not of sufficient population to be observable at atmospheric pressure. Recently, the Hubbell group at the University of California, Los Angeles, reintroduced the application of high pressure to the study of proteins by electron paramagnetic resonance (EPR) spectroscopy. This methodology is possible using X-band EPR spectroscopy due to advances in pressure intensifiers, sample cells, and resonators. In addition to the commercial availability of the pressure generation and sample cells by Pressure Biosciences Inc., a five-loop-four-gap resonator required for the initial high pressure EPR spectroscopy experiments by the Hubbell group, and those reported here, was designed by James S. Hyde and built and modified at the National Biomedical EPR Center. With these technological advances, we determined the effect of pressure on the essential periplasmic lipopolysaccharide (LPS) transport protein from Escherichia coli, LptA, and one of its binding partners, LptC. LptA unfolds from the N-terminus to the C-terminus, binding of LPS does not appreciably stabilize the protein under pressure, and monomeric LptA unfolds somewhat more readily than oligomeric LptA upon pressurization to 2 kbar. LptC exhibits a fold and relative lack of stability upon LPS binding similar to LptA, yet adopts an altered, likely monomeric, folded conformation under pressure with only its C-terminus unraveling. The pressure-induced changes likely correlate with functional changes associated with binding and transport of LPS.

Forkhead box transcription factor FoxC1 preserves corneal transparency by regulating vascular growth.

Normal vision requires the precise control of vascular growth to maintain corneal transparency. Here we provide evidence for a unique mechanism by which the Forkhead box transcription factor FoxC1 regulates corneal vascular development. Murine Foxc1 is essential for development of the ocular anterior segment, and in humans, mutations have been identified in Axenfeld-Rieger syndrome, a disorder characterized by anterior segment dysgenesis. We show that FOXC1 mutations also lead to corneal angiogenesis, and that mice homozygous for either a global (Foxc1(-/-)) or neural crest (NC)-specific (NC-Foxc1(-/-)) null mutation display excessive growth of corneal blood and lymphatic vessels. This is associated with disorganization of the extracellular matrix and increased expression of multiple matrix metalloproteinases. Heterozygous mutants (Foxc1(+/-) and NC-Foxc1(+/-)) exhibit milder phenotypes, such as disrupted limbal vasculature. Moreover, environmental exposure to corneal injury significantly increases growth of both blood and lymphatic vessels in both Foxc1(+/-) and NC-Foxc1(+/-) mice compared with controls. Notably, this amplification of the angiogenic response is abolished by inhibition of VEGF receptor 2. Collectively, these findings identify a role for FoxC1 in inhibiting corneal angiogenesis, thereby maintaining corneal transparency by regulating VEGF signaling.

EPR spectroscopy of MolB2C2-a reveals mechanism of transport for a bacterial type II molybdate importer.

In bacteria, ATP-binding cassette (ABC) transporters are vital for the uptake of nutrients and cofactors. Based on differences in structure and activity, ABC importers are divided into two types. Type I transporters have been well studied and employ a tightly regulated alternating access mechanism. Less is known about Type II importers, but much of what we do know has been observed in studies of the vitamin B12 importer BtuC2D2. MolB2C2 (formally known as HI1470/71) is also a Type II importer, but its substrate, molybdate, is ∼10-fold smaller than vitamin B12. To understand mechanistic differences among Type II importers, we focused our studies on MolBC, for which alternative conformations may be required to transport its relatively small substrate. To investigate the mechanism of MolBC, we employed disulfide cross-linking and EPR spectroscopy. From these studies, we found that nucleotide binding is coupled to a conformational shift at the periplasmic gate. Unlike the larger conformational changes in BtuCD-F, this shift in MolBC-A is akin to unlocking a swinging door: allowing just enough space for molybdate to slip into the cell. The lower cytoplasmic gate, identified in BtuCD-F as "gate I," remains open throughout the MolBC-A mechanism, and cytoplasmic gate II closes in the presence of nucleotide. Combining our results, we propose a peristaltic mechanism for MolBC-A, which gives new insight in the transport of small substrates by a Type II importer.

Sonic hedgehog-modified human CD34+ cells preserve cardiac function after acute myocardial infarction.

RATIONALE: Ischemic cardiovascular disease represents one of the largest epidemics currently facing the aging population. Current literature has illustrated the efficacy of autologous, stem cell therapies as novel strategies for treating these disorders. The CD34+ hematopoetic stem cell has shown significant promise in addressing myocardial ischemia by promoting angiogenesis that helps preserve the functionality of ischemic myocardium. Unfortunately, both viability and angiogenic quality of autologous CD34+ cells decline with advanced age and diminished cardiovascular health. OBJECTIVE: To offset age- and health-related angiogenic declines in CD34+ cells, we explored whether the therapeutic efficacy of human CD34+ cells could be enhanced by augmenting their secretion of the known angiogenic factor, sonic hedgehog (Shh). METHODS AND RESULTS: When injected into the border zone of mice after acute myocardial infarction, Shh-modified CD34+ cells (CD34(Shh)) protected against ventricular dilation and cardiac functional declines associated with acute myocardial infarction. Treatment with CD34(Shh) also reduced infarct size and increased border zone capillary density compared with unmodified CD34 cells or cells transfected with the empty vector. CD34(Shh) primarily store and secrete Shh protein in exosomes and this storage process appears to be cell-type specific. In vitro analysis of exosomes derived from CD34(Shh) revealed that (1) exosomes transfer Shh protein to other cell types, and (2) exosomal transfer of functional Shh elicits induction of the canonical Shh signaling pathway in recipient cells. CONCLUSIONS: Exosome-mediated delivery of Shh to ischemic myocardium represents a major mechanism explaining the observed preservation of cardiac function in mice treated with CD34(Shh) cells.

Geminin is required for mitotic proliferation of spermatogonia.

Spermatogonial stem cells divide throughout life, maintaining their own population and giving rise to differentiated gametes. The unstable regulatory protein Geminin is thought to be one of the factors that determine whether stem cells continue to divide or terminally differentiate. Geminin regulates the extent of DNA replication and is thought to maintain cells in an undifferentiated state by inhibiting various transcription factors and chromatin remodeling proteins. To examine how Geminin might regulate spermatogenesis, we developed two conditional mouse models in which the Geminin gene (Gmnn) is deleted from either spermatogonia or meiotic spermatocytes. Deleting Geminin from spermatogonia causes complete sterility in male mice. Gmnn(-/-) spermatogonia disappear during the initial wave of mitotic proliferation that occurs during the first week of life. Gmnn(-/-) spermatogonia exhibit more double-stranded DNA breaks than control cells, consistent with a defect in DNA replication. They maintain expression of genes associated with the undifferentiated state and do not prematurely express genes characteristic of more differentiated spermatogonia. In contrast, deleting Geminin from spermatocytes does not disrupt meiosis or the differentiation of spermatids into mature sperm. In females, Geminin is not required for meiosis, oocyte differentiation, or fertility after the embryonic period of mitotic proliferation has ceased. We conclude that Geminin is absolutely required for mitotic proliferation of spermatogonia but does not regulate their differentiation. Our results suggest that Geminin maintains replication fidelity during the mitotic phase of spermatogenesis, ensuring the precise duplication of genetic information for transmission to the next generation.

Exosomes from human CD34(+) stem cells mediate their proangiogenic paracrine activity.

RATIONALE: Transplantation of human CD34(+) stem cells to ischemic tissues has been associated with reduced angina, improved exercise time, and reduced amputation rates in phase 2 clinical trials and has been shown to induce neovascularization in preclinical models. Previous studies have suggested that paracrine factors secreted by these proangiogenic cells are responsible, at least in part, for the angiogenic effects induced by CD34(+) cell transplantation. OBJECTIVE: Our objective was to investigate the mechanism of CD34(+) stem cell-induced proangiogenic paracrine effects and to examine if exosomes, a component of paracrine secretion, are involved. METHODS AND RESULTS: Exosomes collected from the conditioned media of mobilized human CD34(+) cells had the characteristic size (40 to 90 nm; determined by dynamic light scattering), cup-shaped morphology (electron microscopy), expressed exosome-marker proteins CD63, phosphatidylserine (flow cytometry) and TSG101 (immunoblotting), besides expressing CD34(+) cell lineage marker protein, CD34. In vitro, CD34(+) exosomes replicated the angiogenic activity of CD34(+) cells by increasing endothelial cell viability, proliferation, and tube formation on Matrigel. In vivo, the CD34(+) exosomes stimulated angiogenesis in Matrigel plug and corneal assays. Interestingly, exosomes from CD34(+) cells but not from CD34(+) cell-depleted mononuclear cells had angiogenic activity. CONCLUSIONS: Our data demonstrate that human CD34(+) cells secrete exosomes that have independent angiogenic activity both in vitro and in vivo. CD34(+) exosomes may represent a significant component of the paracrine effect of progenitor cell transplantation for therapeutic angiogenesis.

Conformational changes in the activation loop of a bacterial PASTA kinase.

Many bacterial genomes encode a transmembrane protein kinase belonging to the PASTA kinase family, which controls numerous processes in diverse bacterial pathogens, including antibiotic resistance, cell division, stress resistance, toxin production, and virulence. PASTA kinases share a conserved three-part domain architecture, consisting of an extracellular PASTA domain, proposed to sense the peptidoglycan layer status, a single transmembrane helix, and an intracellular Ser/Thr kinase domain. The crystal structures of the kinase domain from two homologous PASTA kinases reveal a characteristic two-lobed structure typical of eukaryotic protein kinases with a centrally located, but unresolved, activation loop that becomes phosphorylated and regulates downstream signaling pathways. We previously identified three sites of phosphorylation on the activation loop (T163, T166, and T168) of IreK, a PASTA kinase from the pathogen Enterococcus faecalis, as well as a distal phosphorylation site (T218) that each influence IreK activity in vivo. Still, the mechanism by which loop phosphorylation regulates PASTA kinase function is yet unknown. Therefore, we utilized site-directed spin labeling (SDSL) and continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy to assess the E. faecalis IreK kinase activation loop dynamics, including the effects of phosphorylation on activation loop motion, and the IreK-IreB interaction. Our results reveal that the IreK activation loop occupies a more immobile state when dephosphorylated, and that loop autophosphorylation shifts the loop to a more mobile state that can then enable interaction with IreB, a known substrate.

Multisite Phosphorylation Regulates GpsB Function in Cephalosporin Resistance of Enterococcus faecalis.

Enterococci are normal human commensals and major causes of hospital-acquired infections. Enterococcal infections can be difficult to treat because enterococci harbor intrinsic and acquired antibiotic resistance, such as resistance to cephalosporins. In Enterococcus faecalis, the transmembrane kinase IreK, a member of the bacterial PASTA kinase family, is essential for cephalosporin resistance. The activity of IreK is boosted by the cytoplasmic protein GpsB, which promotes IreK autophosphorylation and signaling to drive cephalosporin resistance. A previous phosphoproteomics study identified eight putative IreK-dependent phosphorylation sites on GpsB, but the functional importance of GpsB phosphorylation was unknown. Here we used genetic and biochemical approaches to define three sites of phosphorylation on GpsB that functionally impact IreK activity and cephalosporin resistance. Phosphorylation at two sites (S80 and T84) serves to impair the ability of GpsB to activate IreK in vivo, suggesting phosphorylation of these sites acts as a means of negative feedback for IreK. The third site of phosphorylation (T133) occurs in a segment of GpsB termed the C-terminal extension that is unique to enterococcal GpsB homologs. The C-terminal extension is highly mobile in solution, suggesting it is largely unstructured, and phosphorylation of T133 appears to enable efficient phosphorylation at S80 / T84. Overall our results are consistent with a model in which multisite phosphorylation of GpsB impairs its ability to activate IreK, thereby diminishing signal transduction through the IreK-dependent pathway and modulating phenotypic cephalosporin resistance.

Binding and transport of LPS occurs through the coordinated combination of an array of sites across the entire Escherichia coli LPS transport protein LptA.

The outer leaflet of the outer membrane (OM) of bacteria such as Escherichia coli, Pseudomonas aeruginosa, and other important pathogens is largely composed of lipopolysaccharide (LPS), which is essential to nearly all Gram-negative bacteria. LPS is transported to the outer leaflet of the OM through a yet unknown mechanism by seven proteins that comprise the LPS transport system. LptA, the only entirely periplasmic Lpt protein, bridges the periplasmic space between the IM LptB2 FGC and the OM LptDE complexes. LptA is postulated to protect the hydrophobic acyl chains of LPS as it crosses the hydrophilic periplasm, is essential to cell viability, and contains many conserved residues distributed across the protein. To identify which side chains are required for function of E. coli LptA in vivo, we performed a systematic, unbiased, high-throughput screen of the effect of 172 single alanine substitutions on cell viability utilizing an engineered BL21 derivative with a chromosomal knockout of the lptA gene. Remarkably, LptA is highly tolerant to amino acid substitution with alanine. Only four alanine mutants could not complement the chromosomal knockout; CD spectroscopy showed that these substitutions resulted in proteins with significantly altered secondary structure. In addition, 29 partial loss-of-function mutants were identified that led to OM permeability defects; interestingly, these sites were solely located within ß-strands of the central core of the protein and each resulted in misfolding of the protein. Therefore, no single residue within LptA is responsible for LPS binding, supporting previous EPR spectroscopy data indicating that sites across the entire protein work in concert to bind and transport LPS.

Generation of conditional alleles for Foxc1 and Foxc2 in mice.

The Forkhead box transcription factors, Foxc1 and Foxc2, are crucial for development of the eye, cardiovascular network, and other physiological systems, but their cell-type specific and postdevelopmental functions are unknown, in part because conventional (i.e., whole-organism) homozygous-null mutations of either factor result in perinatal death. Here, we describe the generation of mice with conditional-null Foxc1(flox) and Foxc2(flox) mutations that are induced via Cre-mediated recombination. Mice homozygous for the unrecombined alleles are viable and fertile, indicating that the conditional alleles retain their wild-type function. The embryos of Foxc1(flox) or Foxc2(flox) mice crossed with Cre-deleter mice that are homozygous for the recombined allele (i.e., Foxc1(Δ/Δ) or Foxc2(Δ/Δ) embryos) lack expression of the corresponding gene and show the same developmental defects observed in conventional homozygous mutant embryos. We expect these conditional mutations to enable characterization of the cell-type specific functions of Foxc1 and Foxc2 in development, disease, and adult animals.

Use of Site-Directed Spin Labeling EPR Spectroscopy to Study Protein-LPS Interactions.

Site-directed spin labeling EPR (electron paramagnetic resonance) spectroscopy is a technique used to identify the local conformational changes at a specific residue of interest within a purified protein in response to a ligand. Here, we describe the site-directed spin labeling EPR spectroscopy methodology to monitor changes in the side-chain motion in soluble lipopolysaccharide transport proteins upon the addition of lipopolysaccharide (LPS). A comparison of the spectral overlays of the spin-labeled protein in the absence and presence of LPS provides a qualitative visualization of how LPS binding affects the motion of each spin-labeled site tested within the protein. No change in the spectral lineshapes of a spin-labeled protein in the absence and presence of LPS indicates that the site is not affected by LPS binding, while differences in the spectral lineshapes indicate that LPS does affect the mobility of the spin label side chain within the protein structure. This is a powerful readout of conformational changes at specific residues of interest that can be used to identify a specific site as a reporter of changes induced by ligand binding and to map out the effects of ligand binding through an array of reporter sites within a protein. With the use of AquaStar tubing, protein concentrations as low as 2 µM allow for up to a 100-fold excess of LPS. This methodology may also be applied to other protein-ligand or protein-protein interactions with minor adaptations.

The Two Non-Visual Arrestins Engage ERK2 Differently.

Arrestin binding to active phosphorylated G protein-coupled receptors terminates G protein coupling and initiates another wave of signaling. Among the effectors that bind directly to receptor-associated arrestins are extracellular signal-regulated kinases 1/2 (ERK1/2), which promote cellular proliferation and survival. Arrestins may also engage ERK1/2 in isolation in a pre- or post-signaling complex that is likely in equilibrium with the full signal initiation complex. Molecular details of these binary complexes remain unknown. Here, we investigate the molecular mechanisms whereby arrestin-2 and arrestin-3 (a.k.a. ß-arrestin1 and ß-arrestin2, respectively) engage ERK1/2 in pairwise interactions. We find that purified arrestin-3 binds ERK2 more avidly than arrestin-2. A combination of biophysical techniques and peptide array analysis demonstrates that the molecular basis in this difference of binding strength is that the two non-visual arrestins bind ERK2 via different parts of the molecule. We propose a structural model of the ERK2-arrestin-3 complex in solution using size-exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS). This binary complex exhibits conformational heterogeneity. We speculate that this drives the equilibrium either toward the full signaling complex with receptor-bound arrestin at the membrane or toward full dissociation in the cytoplasm. As ERK1/2 regulates cell migration, proliferation, and survival, understanding complexes that relate to its activation could be exploited to control cell fate.

Effects of the L511P and D512G mutations on the Escherichia coli ABC transporter MsbA.

MsbA is a member of the ABC transporter superfamily and is homologous to ABC transporters linked to multidrug resistance. The nucleotide binding domains (NBDs) of these proteins include conserved motifs that are involved in ATP binding, including conserved SALD residues (D-loop) that are diagnostic in identifying ABC transporters but whose roles have not been identified. Within the D-loop, single point mutations L511P and D512G were discovered by random mutational analysis of MsbA to disrupt protein function in the cell [Polissi, A., and Georgopoulos, C. (1996) Mol. Microbiol. 20, 1221-1233] but have not been further studied in MsbA or in detail in any other ABC transporter. In these studies, we show that both L511P and D512G mutants of MsbA are able to bind ATP at near-wild-type levels but are unable to maintain cell viability in an in vivo growth assay, verifying the theory that they are dysfunctional at some point after ATP binding. An ATPase assay further suggests that the L511P mutation prevents effective ATP hydrolysis, and an ATP detection assay reveals that only small amounts of ATP are hydrolyzed; D512G is able to hydrolyze ATP at a rate 3-fold faster than that of the wild type. EPR spectroscopy studies using reporter sites within the NBDs also indicate that at least some hydrolysis occurs in L511P or D512G MsbA but show fewer spectral changes than observed for the same reporters in the wild-type background. These studies indicate that L511 is necessary for efficient ATP hydrolysis and D512 is essential for conformational rearrangements required for flipping lipid A.

Characterization of the E506Q and H537A dysfunctional mutants in the E. coli ABC transporter MsbA.

MsbA is a member of the ABC transporter superfamily that is specifically found in Gram-negative bacteria and is homologous to proteins involved in both bacterial and human drug resistance. The E506Q and H537A mutations have been introduced and used for crystallization of other members of the ABC transporter protein family, including BmrA and the ATPase domains MalK, HlyB-NBD, and MJ0796, but have not been previously studied in detail or investigated in the MsbA lipid A exporter. We utilized an array of biochemical and EPR spectroscopy approaches to characterize the local and global effects of these nucleotide binding domain mutations on the E. coli MsbA homodimer. The lack of cell viability in an in vivo growth assay confirms that the presence of the E506Q or H537A mutations within MsbA creates a dysfunctional protein. To further investigate the mode of dysfunction, a fluorescent ATP binding assay was used and showed that both mutant proteins maintain their ability to bind ATP, but ATPase assays indicate hydrolysis is severely inhibited by each mutation. EPR spectroscopy data using previously identified and characterized reporter sites within the nucleotide binding domain along with ATP detection assays show that hydrolysis does occur over time in both mutants, though more readily in the H537A protein. DEER spectroscopy demonstrates that both proteins studied are purified in a closed dimer conformation, indicating that events within the cell can induce a stable, closed conformation of the MsbA homodimer that does not reopen even in the absence of nucleotide.

Characterization of and lipopolysaccharide binding to the E. coli LptC protein dimer.

Lipopolysaccharide (LPS, endotoxin) is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium. LPS is a large lipid containing several acyl chains as its hydrophobic base and numerous sugars as its hydrophilic core and O-antigen domains, and is an essential element of the organisms' natural defenses in adverse environmental conditions. LptC is one of seven members of the lipopolysaccharide transport (Lpt) protein family that functions to transport LPS from the inner membrane (IM) to the outer leaflet of the outer membrane of the bacterium. LptC is anchored to the IM and associated with the IM LptFGB2 complex. It is hypothesized that LPS binds to LptC at the IM, transfers to LptA to cross the periplasm, and is inserted by LptDE into the outer leaflet of the outer membrane. The studies described here comprehensively characterize and quantitate the binding of LPS to LptC. Site-directed spin labeling electron paramagnetic resonance spectroscopy was utilized to characterize the LptC dimer in solution and monitor spin label mobility changes at 10 sites across the protein upon addition of exogenous LPS. The results indicate that soluble LptC forms concentration-independent N-terminal dimers in solution, LptA binding does not change the conformation of the LptC dimer nor appreciably disrupt the LptC dimer in vitro, and LPS binding affects the entire LptC protein, with the center and C-terminal regions showing a greater affinity for LPS than the N-terminal domain, which has similar dissociation constants to LptA.

Disruption of the E. coli LptC dimerization interface and characterization of lipopolysaccharide and LptA binding to monomeric LptC.

Lipopolysaccharide (LPS) is an essential element of nearly all Gram-negative bacterial outer membranes and serves to protect the cell from adverse environmental stresses. Seven members of the lipopolysaccharide transport (Lpt) protein family function together to transport LPS from the inner membrane (IM) to the outer leaflet of the outer membrane of bacteria such as Escherichia coli. Each of these proteins has a solved crystal structure, including LptC, which is a largely periplasmic protein that is associated with the IM LptB2 FG complex and anchored to the membrane by an N-terminal helix. LptC directly binds LPS and is hypothesized to be involved in the transfer of LPS to another periplasmic protein, LptA. Purified and in solution, LptC forms a dimer. Here, point mutations designed to disrupt formation of the dimer are characterized using site-directed spin labeling double electron electron resonance (DEER) spectroscopy, light scattering, circular dichroism, and computational modeling. The computational studies reveal the molecular interactions that drive dimerization of LptC and elucidate how the disruptive mutations change this interaction, while the DEER and light scattering studies identify which mutants disrupt the dimer. And, using electron paramagnetic resonance spectroscopy and comparing the results to the previous quantitative characterization of the interactions between dimeric LptC and LPS and LptA, the functional consequences of monomeric LptC were also determined. These results indicate that disruption of the dimer does not affect LPS or LptA binding and that monomeric LptC binds LPS and LptA at levels similar to dimeric LptC.

Lipopolysaccharide binding to the periplasmic protein LptA.

Lipopolysaccharide (LPS) and the periplasmic protein, LptA, are two essential components of Gram-negative bacteria. LPS, also known as endotoxin, is found asymmetrically distributed in the outer leaflet of the outer membrane of Gram-negative bacteria such as Escherichia coli and plays a role in the organism's natural defense in adverse environmental conditions. LptA is a member of the lipopolysaccharide transport protein (Lpt) family, which also includes LptC, LptDE, and LptBFG2 , that functions to transport LPS through the periplasm to the outer leaflet of the outer membrane after MsbA flips LPS across the inner membrane. It is hypothesized that LPS binds to LptA to cross the periplasm and that the acyl chains of LPS bind to the central pocket of LptA. The studies described here are the first to comprehensively characterize and quantitate the binding of LPS by LptA. Using site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy, data were collected for 15 spin-labeled residues in and around the proposed LPS binding pocket on LptA to observe the mobility changes caused by the presence of exogenous LPS and identify the binding location of LPS to LptA. The EPR data obtained suggest a 1:1 ratio for the LPS:LptA complex and allow the first calculation of dissociation constants for the LptA-LPS interaction. The results indicate that the entire protein is affected by LPS binding, the N-terminus unfolds in the presence of LPS, and a mutant LptA protein unable to form oligomers has an altered affinity for LPS.

Foxc1 and Foxc2 in the Neural Crest Are Required for Ocular Anterior Segment Development.

Purpose: The large Forkhead (Fox) transcription factor family has essential roles in development, and mutations cause a wide range of ocular and nonocular disease. One member, Foxc2 is expressed in neural crest (NC)-derived periocular mesenchymal cells of the developing murine eye; however, its precise role in the development, establishment, and maintenance of the ocular surface has yet to be investigated. Methods: To specifically delete Foxc2 from NC-derived cells, conditional knockout mice for Foxc2 (NC-Foxc2-/-) were generated by crossing Foxc2F mice with Wnt1-Cre mice. Similarly, we also generated compound NC-specific mutations of Foxc2 and a closely related gene, Foxc1 (NC-Foxc1-/-;NC-Foxc2-/-) in mice. Results: Neural crest-Foxc2-/- mice show abnormal thickness in the peripheral-to-central corneal stroma and limbus and displaced pupils with irregular iris. The neural crest-specific mutation in Foxc2 also leads to ectopic neovascularization in the cornea, as well as impaired ocular epithelial cell identity and corneal conjunctivalization. Compound, NC-specific Foxc1; Foxc2 homozygous mutant mice have more severe defects in structures of the ocular surface, such as the cornea and eyelids, accompanied by significant declines in the expression of another key developmental factor, Pitx2, and its downstream effector Dkk2, which antagonizes canonical Wnt signaling. Conclusions: The neural crest-Foxc2 mutation is associated with corneal conjunctivalization, ectopic corneal neovascularization, and disrupted ocular epithelial cell identity. Furthermore, Foxc2 and Foxc1 cooperatively function in NC-derived mesenchymal cells to ensure proper morphogenesis of the ocular surface via the regulation of Wnt signaling. Together, Foxc2 is required in the NC lineage for mesenchymal-epithelial interactions in corneal and ocular surface development.

Directed evolution provides insight into conformational substrate sampling by SrtA.

The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest for the development of novel anti-microbials, however, more recently their function as unique transpeptidases has been exploited for the synthesis of novel bio-conjugates. Yet, for synthetic purposes, SrtA-mediated conjugation suffers from the enzyme's inherently poor catalytic efficiency. Therefore, to identify SrtA variants with improved catalytic efficiency, we used directed evolution to select a catalytically enhanced SrtA enzyme. An analysis of improved SrtA variants in the context of sequence conservation, NMR and x-ray crystal structures, and kinetic data suggests a novel mechanism for catalysis involving large conformational changes that delivers substrate to the active site pocket. Indeed, using DEER-EPR spectroscopy, we reveal that upon substrate binding, SrtA undergoes a large scissors-like conformational change that simultaneously translates the sort-tag substrate to the active site in addition to repositioning key catalytic residues for esterification. A better understanding of Sortase dynamics will significantly enhance future engineering and drug discovery efforts.

Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation.

Polymyxins are antibiotics used in the last line of defense to combat multidrug-resistant infections by Gram-negative bacteria. Polymyxin resistance arises through charge modification of the bacterial outer membrane with the attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose to lipid A, a reaction catalyzed by the integral membrane lipid-to-lipid glycosyltransferase 4-amino-4-deoxy-L-arabinose transferase (ArnT). Here, we report crystal structures of ArnT from Cupriavidus metallidurans, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.8 and 3.2 angstrom resolution, respectively. The structures show cavities for both lipidic substrates, which converge at the active site. A structural rearrangement occurs on undecaprenyl phosphate binding, which stabilizes the active site and likely allows lipid A binding. Functional mutagenesis experiments based on these structures suggest a mechanistic model for ArnT family enzymes.