Disulfi de constrained Fabs overcome target size limitation for high-resolution single-particle cryo-EM.

High-resolution structures of proteins are critical to understanding molecular mechanisms of biological processes and in the discovery of therapeutic molecules. Cryo-EM has revolutionized structure determination of large proteins and their complexes1, but a vast majority of proteins that underlie human diseases are small (< 50 kDa) and usually beyond its reach due to low signal-to-noise images and difficulties in particle alignment2. Current strategies to overcome this problem increase the overall size of small protein targets using scaffold proteins that bind to the target, but are limited by inherent flexibility and not being bound to their targets in a rigid manner, resulting in the target being poorly resolved compared to the scaffolds3-11. Here we present an iteratively engineered molecular design for transforming Fabs (antibody fragments), into conformationally rigid scaffolds (Rigid-Fabs) that, when bound to small proteins (~20 kDa), can enable high-resolution structure determination using cryo-EM. This design introduces multiple disulfide bonds at strategic locations, generates a well-folded Fab constrained into a rigid conformation and can be applied to Fabs from various species, isotypes and chimeric Fabs. We present examples of the Rigid Fab design enabling high-resolution (2.3-2.5 Å) structures of small proteins, Ang2 (26 kDa) and KRAS (21 kDa) by cryo-EM. The strategies for designing disulfide constrained Rigid Fabs in our work thus establish a general approach to overcome the target size limitation of single particle cryo-EM.

The formation of tonalitic and granodioritic melt from Venusian basalt.

The crust of Venus is composed of the low lying volcanic planitiae and the elevated, deformed tesserae. It is thought that the tesserae may be composed of silicic igneous rocks and that it may resemble proto-continental crust. The initial development of terrestrial continental crust is likely due to melting and deformation of primitive mafic crust via mantle-plume upwelling and collisional plate processes. Unlike Earth, the lithosphere of Venus is not divided into plates and therefore evolved continental crust, if present, developed primarily by melting of pre-existing mafic crust. Here, we report the results of high pressure equilibrium partial melting experiments using a parental composition similar to the basalt measured at the Venera 14 landing site in order to determine if silicic melts can be generated. It was found that at pressures of 1.5 GPa and 2.0 GPa and temperatures of 1080 °C, 1090 °C, and 1285 °C that tonalitic and granodioritic melts can be generated. The experimental results indicate that silicic rocks may be able to form in the crust of Venus providing the thermal regime is suitable and that the lower crust is basaltic. The implication is that the older, thicker regions of Venusian crust may be partially composed of silicic igneous rocks.

Structural basis for ALK2/BMPR2 receptor complex signaling through kinase domain oligomerization.

Upon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.

PEAK3/C19orf35 pseudokinase, a new NFK3 kinase family member, inhibits CrkII through dimerization.

Members of the New Kinase Family 3 (NKF3), PEAK1/SgK269 and Pragmin/SgK223 pseudokinases, have emerged as important regulators of cell motility and cancer progression. Here, we demonstrate that C19orf35 (PEAK3), a newly identified member of the NKF3 family, is a kinase-like protein evolutionarily conserved across mammals and birds and a regulator of cell motility. In contrast to its family members, which promote cell elongation when overexpressed in cells, PEAK3 overexpression does not have an elongating effect on cell shape but instead is associated with loss of actin filaments. Through an unbiased search for PEAK3 binding partners, we identified several regulators of cell motility, including the adaptor protein CrkII. We show that by binding to CrkII, PEAK3 prevents the formation of CrkII-dependent membrane ruffling. This function of PEAK3 is reliant upon its dimerization, which is mediated through a split helical dimerization domain conserved among all NKF3 family members. Disruption of the conserved DFG motif in the PEAK3 pseudokinase domain also interferes with its ability to dimerize and subsequently bind CrkII, suggesting that the conformation of the pseudokinase domain might play an important role in PEAK3 signaling. Hence, our data identify PEAK3 as an NKF3 family member with a unique role in cell motility driven by dimerization of its pseudokinase domain.

Prospects for pharmacological targeting of pseudokinases.

Pseudokinases are members of the protein kinase superfamily but signal primarily through noncatalytic mechanisms. Many pseudokinases contribute to the pathologies of human diseases, yet they remain largely unexplored as drug targets owing to challenges associated with modulation of their biological functions. Our understanding of the structure and physiological roles of pseudokinases has improved substantially over the past decade, revealing intriguing similarities between pseudokinases and their catalytically active counterparts. Pseudokinases often adopt conformations that are analogous to those seen in catalytically active kinases and, in some cases, can also bind metal cations and/or nucleotides. Several clinically approved kinase inhibitors have been shown to influence the noncatalytic functions of active kinases, providing hope that similar properties in pseudokinases could be pharmacologically regulated. In this Review, we discuss known roles of pseudokinases in disease, their unique structural features and the progress that has been made towards developing pseudokinase-directed therapeutics.

The pseudokinase TRIB1 toggles an intramolecular switch to regulate COP1 nuclear export.

COP1 is a highly conserved ubiquitin ligase that regulates diverse cellular processes in plants and metazoans. Tribbles pseudokinases, which only exist in metazoans, act as scaffolds that interact with COP1 and its substrates to facilitate ubiquitination. Here, we report that, in addition to this scaffolding role, TRIB1 promotes nuclear localization of COP1 by disrupting an intramolecular interaction between the WD40 domain and a previously uncharacterized regulatory site within COP1. This site, which we have termed the pseudosubstrate latch (PSL), resembles the consensus COP1-binding motif present in known COP1 substrates. Our findings support a model in which binding of the PSL to the WD40 domain stabilizes a conformation of COP1 that is conducive to CRM1-mediated nuclear export, and TRIB1 displaces this intramolecular interaction to induce nuclear retention of COP1. Coevolution of Tribbles and the PSL in metazoans further underscores the importance of this role of Tribbles in regulating COP1 function.

Management of Complex Epithelial Ingrowth After Laser In Situ Keratomileusis Using Fibrin Tissue Glue.

OBJECTIVES: To evaluate the efficacy and safety of adjunctive fibrin tissue glue in the treatment of complex epithelial ingrowth after laser in situ keratomileusis (LASIK). METHODS: A retrospective review was performed of 12 eyes in 12 patients treated for clinically significant epithelial ingrowth after LASIK with mechanical debridement of the ingrowth and placement of fibrin tissue glue. Primary outcome measurements including recurrence of ingrowth, visual acuity, and manifest refraction were evaluated at each postoperative examination. Changes in higher-order aberrometry were also evaluated. RESULTS: After epithelial ingrowth removal with adjunctive fibrin tissue glue, 11 eyes (91.7%) had no recurrence of ingrowth at the final follow-up examination. Uncorrected distance visual acuity changed from 20/20 or better in 3 eyes (25%) and 20/40 or better in 6 eyes (50%) preoperatively to 20/20 or better in 5 eyes (41.7%) and 20/40 or better in 10 eyes (83.3%) postoperatively. Nine eyes (75%) gained one or more lines of best-corrected distance visual acuity (CDVA). No eyes lost any lines of CDVA. There was no significant change in mean spherical equivalent (P=0.22) or mean cylinder (P=0.26) before and after surgery. Higher-order aberrations also remained stable with no significant change in root-mean-square error, coma, trefoil, and spherical aberration. There were no complications associated with the treatment. CONCLUSIONS: Adjunctive fibrin tissue glue seems to be a safe and effective treatment for epithelial ingrowth after LASIK. This therapy may be particularly useful in managing challenging cases of complex or recalcitrant ingrowth.

Deafferented Adult Rod Bipolar Cells Create New Synapses with Photoreceptors to Restore Vision.

Upon degeneration of photoreceptors in the adult retina, interneurons, including bipolar cells, exhibit a plastic response leading to their aberrant rewiring. Photoreceptor reintroduction has been suggested as a potential approach to sight restoration, but the ability of deafferented bipolar cells to establish functional synapses with photoreceptors is poorly understood. Here we use photocoagulation to selectively destroy photoreceptors in adult rabbits while preserving the inner retina. We find that rods and cones shift into the ablation zone over several weeks, reducing the blind spot at scotopic and photopic luminances. During recovery, rod and cone bipolar cells exhibit markedly different responses to deafferentation. Rod bipolar cells extend their dendrites to form new synapses with healthy photoreceptors outside the lesion, thereby restoring visual function in the deafferented retina. Secretagogin-positive cone bipolar cells did not exhibit such obvious dendritic restructuring. These findings are encouraging to the idea of photoreceptor reintroduction for vision restoration in patients blinded by retinal degeneration. At the same time, they draw attention to the postsynaptic side of photoreceptor reintroduction; various bipolar cell types, representing different visual pathways, vary in their response to the photoreceptor loss and in their consequent dendritic restructuring.SIGNIFICANCE STATEMENT Loss of photoreceptors during retinal degeneration results in permanent visual impairment. Strategies for vision restoration based on the reintroduction of photoreceptors inherently rely on the ability of the remaining retinal neurons to correctly synapse with new photoreceptors. We show that deafferented bipolar cells in the adult mammalian retina can reconnect to rods and cones and restore retinal sensitivity at scotopic and photopic luminances. Rod bipolar cells extend their dendrites to form new synapses with healthy rod photoreceptors. These findings support the idea that bipolar cells might be able to synapse with reintroduced photoreceptors, thereby restoring vision in patients blinded by retinal degeneration.

Correction: A highly flexible inorganic framework with amphiphilic amine assemblies as templates.

Correction for 'A highly flexible inorganic framework with amphiphilic amine assemblies as templates' by Hui-Lin Huang et al., Dalton Trans., 2017, DOI: 10.1039/c6dt04165e.

A highly flexible inorganic framework with amphiphilic amine assemblies as templates.

A zinc phosphite-phosphate framework, (HA)2[Zn3(HPO3)4-x(HPO4)x] (1; A = cha, coa, iba, pa, and ha; x = 0.3-1) with nanometer-scale channels was prepared. The framework exhibited an extraordinarily high flexibility, 13% expansion at ambient pressure and 27% contraction under a high pressure of over 2.3 GPa without undergoing any phase transformation. The volume changes in the compression-decompression process are reversible. Such unusual adaptability is rare in pure inorganic networks. The molecular volumes of templates range from 165 Å3 to 228 Å3, which allows to vary channel aperture increasingly from 13.02 to 15.34 Å. Remarkably, three types of organic amine template assemblies, captured in inorganic frameworks, were identified in this study. Presented herein is the first example that demonstrates a successfully controlled template assembly that helps to obtain a flexible inorganic framework with nanosized pores in a systematic manner.

Quality of Vision After Wavefront-Guided or Wavefront-Optimized LASIK: A Prospective Randomized Contralateral Eye Study.

PURPOSE: To compare the effect of wavefront-guided and wavefront-optimized LASIK using different laser platforms on subjective quality of vision. METHODS: The dominant eyes of 55 participants with myopia were randomized to receive either wavefront-guided LASIK treatment by the VISX Star S4 IR Custom-Vue excimer laser system (Abbott Medical Optics, Inc., Santa Clara, CA) or wavefront-optimized treatment by the WaveLight Allegretto Wave Eye-Q 400-Hz excimer laser system (Alcon Laboratories, Inc., Fort Worth, TX), whereas the fellow eye had the alternate laser treatment. Patients completed a questionnaire assessing quality of vision and visual symptoms (daytime and nighttime glare, daytime and nighttime clarity, halos, haze, fluctuating vision, and double vision) preoperatively and at postoperative months 1, 3, 6, and 12. RESULTS: At 3, 6, and 12 months postoperatively, there was no significant difference in any individual symptom between the wavefront-guided and wavefront-optimized groups, although at 12 months wavefront-guided eyes trended toward having more excellent vision (wavefront-guided vs wavefront-optimized; 2.26 vs 2.43; P = .039). In the subgroup of patients with preoperative root mean square (RMS) higher order aberrations (HOAs) less than 0.3 µm in both eyes, the wavefront-optimized group demonstrated a trend toward worsened nighttime clarity (P = .009), daytime clarity (P = .015), and fluctuating vision (P = .046), and less excellent vision (P = .009) at 12 months. CONCLUSIONS: Twelve months after surgery, most patients' self-reported visual symptoms were similar in eyes receiving wavefront-guided or wavefront-optimized LASIK. In general, 36% of patients preferred wavefront-guided LASIK, 19% preferred wavefront-optimized LASIK, and 45% had no preference at 12 months. The wavefront-guided preference was more pronounced in patients with lower baseline HOAs (RMS < 0.3 µm).

Structural Basis for the Non-catalytic Functions of Protein Kinases.

Protein kinases are known primarily for their ability to phosphorylate protein substrates, which constitutes an essential biological process. Recently, compelling evidence has accumulated that the functions of many protein kinases extend beyond phosphorylation and include an impressive spectrum of non-catalytic roles, such as scaffolding, allosteric regulation, or even protein-DNA interactions. How the conserved kinase fold shared by all metazoan protein kinases can accomplish these diverse tasks in a specific and regulated manner is poorly understood. In this review, we analyze the molecular mechanisms supporting phosphorylation-independent signaling by kinases and attempt to identify common and unique structural characteristics that enable kinases to perform non-catalytic functions. We also discuss how post-translational modifications, protein-protein interactions, and small molecules modulate these non-canonical kinase functions. Finally, we highlight current efforts in the targeted design of small-molecule modulators of non-catalytic kinase functions, a new pharmacological challenge for which structural considerations are more important than ever.

The FOXO1 Transcription Factor Instructs the Germinal Center Dark Zone Program.

The pathways regulating formation of the germinal center (GC) dark zone (DZ) and light zone (LZ) are unknown. In this study we show that FOXO1 transcription factor expression was restricted to the GC DZ and was required for DZ formation, since its absence in mice led to the loss of DZ gene programs and the formation of LZ-only GCs. FOXO1-negative GC B cells displayed normal somatic hypermutation but defective affinity maturation and class switch recombination. The function of FOXO1 in sustaining the DZ program involved the trans-activation of the chemokine receptor CXCR4, and cooperation with the BCL6 transcription factor in the trans-repression of genes involved in immune activation, DNA repair, and plasma cell differentiation. These results also have implications for the role of FOXO1 in lymphomagenesis because they suggest that constitutive FOXO1 activity might be required for the oncogenic activity of deregulated BCL6 expression.

Development of Animal Models of Local Retinal Degeneration.

PURPOSE: Development of nongenetic animal models of local retinal degeneration is essential for studies of retinal pathologies, such as chronic retinal detachment or age-related macular degeneration. We present two different methods to induce a highly localized retinal degeneration with precise onset time, that can be applied to a broad range of species in laboratory use. METHODS: A 30-µm thin polymer sheet was implanted subretinally in wild-type (WT) rats. The effects of chronic retinal separation from the RPE were studied using histology and immunohistochemistry. Another approach is applicable to species with avascular retina, such as rabbits, where the photoreceptors and RPE were thermally ablated over large areas, using a high power scanning laser. RESULTS: Photoreceptors above the subretinal implant in rats degenerated over time, with 80% of the outer nuclear layer disappearing within a month, and the rest by 3 months. Similar loss was obtained by selective photocoagulation with a scanning laser. Cells in the inner nuclear layer and ganglion cell layer were preserved in both cases. However, there were signs of rewiring and decrease in the size of the bipolar cell terminals in the damaged areas. CONCLUSIONS: Both methods induce highly reproducible degeneration of photoreceptors over a defined area, with complete preservation of the inner retinal neurons during the 3-month follow-up. They provide a reliable platform for studies of local retinal degeneration and development of therapeutic strategies in a wide variety of species.

Cataract surgery in the glaucoma patient.

To summarize the role of cataract surgery in the glaucoma patient, in terms of the effect on intraocular pressure (IOP) as well as diagnostic and therapeutic considerations for those with both conditions. Recent evidence suggests that cataract extraction may produce a significant and sustained IOP reduction in individuals with open-angle glaucoma, ocular hypertension, and angle-closure glaucoma. Cataract removal may improve the practitioner's ability to interpret perimetric testing, and re-establishing perimetric and optic nerve imaging baselines is recommended after cataract surgery. The sequence of cataract surgery relative to glaucoma surgery impacts the likelihood of complications and surgical success. There are multiple benefits to perform cataract surgery prior to glaucoma surgery while cataract surgery after trabeculectomy increases the risk of subsequent filtration failure. As "minimally invasive glaucoma surgeries" continue to improve in terms of efficacy, there is an evolving role for combined cataract and glaucoma surgery in patients with early to moderate stages of glaucoma.

West African Crystalline Maculopathy in Sickle Cell Retinopathy.

Purpose. To describe the first reported case of West African crystalline maculopathy (WACM) from a member of the Benin tribe and explore the association with sickle cell retinopathy. Methods. Full ophthalmic examination and high-resolution ocular coherence tomographic imaging. Patients. 61-year-old patient from an academic retina practice. Results. The patient demonstrated bilateral yellow-green birefringent crystals localized to the inner retina on optical coherence tomography, as well as sickle cell-related neovascularization in the right eye. She reported no consumption of kola nuts. Conclusions. Associated retinal vascular disease may be important in the pathogenesis of crystalline maculopathy.

Corneal sensation and dry eye symptoms after conventional versus inverted side-cut femtosecond LASIK: a prospective randomized study.

PURPOSE: To compare corneal sensation and self-reported dry eye symptoms after femtosecond-assisted LASIK with conventional versus inverted side cuts. DESIGN: Prospective, randomized, eye-to-eye study. PARTICIPANTS: A total of 120 eyes in 60 participants with myopia. METHODS: Fellow eyes were randomized to receive femtosecond-assisted LASIK with a conventional 70-degree side cut made with the 60 kHz IntraLase FS (Abbott Medical Optics, Santa Ana, CA) or an inverted 130-degree side cut made with the 150 kHz IntraLase iFS (Abbott Medical Optics). Cochet-Bonnet aesthesiometry (Luneau Ophthalmologia, Chartes, France) measured corneal sensation (60 mm = normal sensation; <60 mm = depressed sensation) preoperatively and at postoperative months 1, 3, 6, and 12. Participants also completed serial dry eye surveys using the same subscales (frequency, severity, bothersome) as the validated, Rasch-tested, linear-scaled Quality of Vision questionnaire. MAIN OUTCOME MEASURES: Objective corneal sensation and self-reported light sensitivity, dryness, foreign body sensation, and pain/discomfort. RESULTS: Preoperative corneal sensation as measured by mean Cochet-Bonnet aethesiometry was equal between the inverted and conventional side cut groups but was better in eyes with an inverted side cut compared with a conventional side cut at all postoperative months (inverted vs. conventional: 1 month, 14.5 vs. 13.2 mm; 3 months, 24.9 vs. 18.4 mm; 6 months, 51.2 vs. 42.6 mm; 12 months, 59.8 vs. 58.3 mm; all P ≤ 0.02). None of the subjective dry eye parameters demonstrated statistically significant differences between the groups at any time point. CONCLUSIONS: The LASIK flaps with an inverted side cut are associated with superior recovery of corneal sensation compared with flaps with a conventional side cut during the first postoperative year; however, this may not translate to significant improvements in subjective dry eye symptoms.

Crystal structure of Vδ1 T cell receptor in complex with CD1d-sulfatide shows MHC-like recognition of a self-lipid by human γδ T cells.

The nature of the antigens recognized by γδ T cells and their potential recognition of major histocompatibility complex (MHC)-like molecules has remained unclear. Members of the CD1 family of lipid-presenting molecules are suggested ligands for Vδ1 TCR-expressing γδ T cells, the major γδ lymphocyte population in epithelial tissues. We crystallized a Vδ1 TCR in complex with CD1d and the self-lipid sulfatide, revealing the unusual recognition of CD1d by germline Vδ1 residues spanning all complementarity-determining region (CDR) loops, as well as sulfatide recognition separately encoded by nongermline CDR3δ residues. Binding and functional analysis showed that CD1d presenting self-lipids, including sulfatide, was widely recognized by gut Vδ1+ γδ T cells. These findings provide structural demonstration of MHC-like recognition of a self-lipid by γδ T cells and reveal the prevalence of lipid recognition by innate-like T cell populations.

The molecular basis for recognition of CD1d/α-galactosylceramide by a human non-Vα24 T cell receptor.

CD1d-mediated presentation of glycolipid antigens to T cells is capable of initiating powerful immune responses that can have a beneficial impact on many diseases. Molecular analyses have recently detailed the lipid antigen recognition strategies utilized by the invariant Vα24-Jα18 TCR rearrangements of iNKT cells, which comprise a subset of the human CD1d-restricted T cell population. In contrast, little is known about how lipid antigens are recognized by functionally distinct CD1d-restricted T cells bearing different TCRα chain rearrangements. Here we present crystallographic and biophysical analyses of α-galactosylceramide (α-GalCer) recognition by a human CD1d-restricted TCR that utilizes a Vα3.1-Jα18 rearrangement and displays a more restricted specificity for α-linked glycolipids than that of iNKT TCRs. Despite having sequence divergence in the CDR1α and CDR2α loops, this TCR employs a convergent recognition strategy to engage CD1d/αGalCer, with a binding affinity (∼2 µM) almost identical to that of an iNKT TCR used in this study. The CDR3α loop, similar in sequence to iNKT-TCRs, engages CD1d/αGalCer in a similar position as that seen with iNKT-TCRs, however fewer actual contacts are made. Instead, the CDR1α loop contributes important contacts to CD1d/αGalCer, with an emphasis on the 4'OH of the galactose headgroup. This is consistent with the inability of Vα24- T cells to respond to α-glucosylceramide, which differs from αGalCer in the position of the 4'OH. These data illustrate how fine specificity for a lipid containing α-linked galactose is achieved by a TCR structurally distinct from that of iNKT cells.