The alarmone ppGpp selectively inhibits the isoform A of the human small GTPase Sar1.

Transport of newly synthesized proteins from endoplasmic reticulum (ER) to Golgi is mediated by coat protein complex II (COPII). The assembly and disassembly of COPII vesicles is regulated by the molecular switch Sar1, which is a small GTPase and a component of COPII. Usually a small GTPase binds GDP (inactive form) or GTP (active form). Mammals have two Sar1 isoforms, Sar1a and Sar1b, that have approximately 90% sequence identity. Some experiments demonstrated that these two isoforms had distinct but overlapping functions. Here we found another instance of differing behavior: the alarmone ppGpp could bind to and inhibit the GTPase activity of human Sar1a but could not inhibit the GTPase activity of human Sar1b. The crystal structures of Sar1a⋅ppGpp and Sar1b⋅GDP have been determined. Superposition of the structures shows that ppGpp binds to the nucleotide-binding pocket, its guanosine base, ribose ring and 5'-diphosphate occupying nearly the same positions as for GDP. However, its 3'-diphosphate points away from the active site and, hence, away from the surface of the protein. The overall structure of Sar1a⋅ppGpp is more similar to Sar1b⋅GDP than to Sar1b⋅GTP. We also find that the Asp140-Arg138-water-ligand interaction net is important for the binding of ppGpp to Sar1a. This study provides further evidence showing that there are biochemical differences between the Sar1a and Sar1b isoforms of Sar1.

Crystal structure of a type III Rubisco in complex with its product 3-phosphoglycerate.

The crystal structure of the complex of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from Archaeoglobus fulgidus (afRubisco) with its products 3PGAs has been determined to a resolution of 1.7 Å and is of the closed form. Type III Rubiscos such as afRubisco have 18 out of the 19 essential amino acid residues of canonical Rubisco; the 19th is Tyr rather than Phe. Superposition with the structure of a complex of the similar tkRubisco with the six-carbon intermediate analog 2CABP shows the same conformation of the 19 residues except for Glu46 and Thr51. Glu46 adopts a unique conformation different from that in other Rubiscos and makes two H-bonds with the ligand 3PGA. Similar to other closed state Rubiscos, the backbone of Thr51 is rotated and the side chain makes an H-bond with the ligand 3PGA. Two product 3PGA molecules are bound at the active site, overlapping well with the 2CABP of tkRubisco/2CABP. The positions of the P1 and P2 phosphate groups differ by 0.4 and 0.53 Å, respectively, between 2CABP and the two 3PGAs. This afRubisco/3PGA complex mimics an intermediate stage of the carboxylation reaction which occurs after the production of the two 3PGA products but before the reopening of the active site. The stability of this complex suggests that the Rubisco active site will not reopen before both 3PGA products are formed.

Unexpected roles for ADH1 and SORD in catalyzing the final step of erythritol biosynthesis.

The low-calorie sweetener erythritol is endogenously produced from glucose through the pentose phosphate pathway in humans. Erythritol is of medical interest because elevated plasma levels of this polyol are predictive for visceral adiposity gain and development of type 2 diabetes. However, the mechanisms behind these associations remain unknown because the erythritol biosynthesis pathway, particularly the enzyme catalyzing the final step of erythritol synthesis (reduction of erythrose to erythritol), is not characterized. In this study, we purified two enzymes from rabbit liver capable of catalyzing the conversion of erythrose to erythritol: alcohol dehydrogenase 1 (ADH1) and sorbitol dehydrogenase (SORD). Both recombinant human ADH1 and SORD reduce erythrose to erythritol, using NADPH as a co-factor, and cell culture studies indicate that this activity is primarily NADPH-dependent. We found that ADH1 variants vary markedly in both their affinity for erythrose and their catalytic capacity (turnover number). Interestingly, the recombinant protein produced from the ADH1B2 variant, common in Asian populations, is not active when NADPH is used as a co-factor in vitro We also confirmed SORD contributes to intracellular erythritol production in human A549 lung cancer cells, where ADH1 is minimally expressed. In summary, human ADH1 and SORD catalyze the conversion of erythrose to erythritol, pointing to novel roles for two dehydrogenase proteins in human glucose metabolism that may contribute to individual responses to diet. Proteomics data are available via ProteomeXchange with identifier PXD015178.

Structural basis for the interaction between the growth factor-binding protein GRB10 and the E3 ubiquitin ligase NEDD4.

In addition to inhibiting insulin receptor and IGF1R kinase activity by directly binding to the receptors, GRB10 can also negatively regulate insulin and IGF1 signaling by mediating insulin receptor and IGF1R degradation through ubiquitination. It has been shown that GRB10 can interact with the C2 domain of the E3 ubiquitin ligase NEDD4 through its Src homology 2 (SH2) domain. Therefore, GRB10 might act as a connector, bringing NEDD4 close to IGF1R to facilitate the ubiquitination of IGF1R by NEDD4. This is the first case in which it has been found that an SH2 domain could colocalize a ubiquitin ligase and its substrate. Here we report the crystal structure of the NEDD4 C2-GRB10 SH2 complex at 2.0 Å. The structure shows that there are three interaction interfaces between NEDD4 C2 and GRB10 SH2. The main interface centers on an antiparallel ß-sheet composed of the F ß-strand of GRB10 SH2 and the C ß-strand of NEDD4 C2. NEDD4 C2 binds at nonclassical sites on the SH2 domain surface, far from the classical phosphotyrosine-binding pocket. Hence, this interaction is phosphotyrosine-independent, and GRB10 SH2 can bind the C2 domain of NEDD4 and the kinase domain of IGF1R simultaneously. Based on these results, a model of how NEDD4 interacts with IGF1R through GRB10 has been proposed. This report provides further evidence that SH2 domains can participate in important signaling interactions beyond the classical recognition of phosphotyrosine.

Microcrystallography, high-pressure cryocooling and BioSAXS at MacCHESS.

The Macromolecular Diffraction Facility at the Cornell High Energy Synchrotron Source (MacCHESS) is a national research resource supported by the National Center for Research Resources of the US National Institutes of Health. MacCHESS is pursuing several research initiatives designed to benefit both CHESS users and the wider structural biology community. Three initiatives are presented in further detail: microcrystallography, which aims to improve the collection of diffraction data from crystals a few micrometers across, or small well diffracting regions of inhomogeneous crystals, so as to obtain high-resolution structures; pressure cryocooling, which can stabilize transient structures and reduce lattice damage during the cooling process; and BioSAXS (small-angle X-ray scattering on biological solutions), which can extract molecular shape and other structural information from macromolecules in solution.

Feasibility of one-shot-per-crystal structure determination using Laue diffraction.

Crystal size is an important factor in determining the number of diffraction patterns which may be obtained from a protein crystal before severe radiation damage sets in. As crystal dimensions decrease this number is reduced, eventually falling to one, at which point a complete data set must be assembled using data from multiple crystals. When only a single exposure is to be collected from each crystal, the polychromatic Laue technique may be preferable to monochromatic methods owing to its simultaneous recording of a large number of fully recorded reflections per image. To assess the feasibility of solving structures using single Laue images from multiple crystals, data were collected using a 'pink' beam at the CHESS D1 station from groups of lysozyme crystals with dimensions of the order of 20-30 microm mounted on MicroMesh grids. Single-shot Laue data were used for structure determination by molecular replacement and correct solutions were obtained even when as few as five crystals were used.

Insights into the mode of action of a putative zinc transporter CzrB in Thermus thermophilus.

The crystal structures of the cytoplasmic domain of the putative zinc transporter CzrB in the apo and zinc-bound forms reported herein are consistent with the protein functioning in vivo as a homodimer. NMR, X-ray scattering, and size-exclusion chromatography provide support for dimer formation. Full-length variants of CzrB in the apo and zinc-loaded states were generated by homology modeling with the Zn2+/H+ antiporter YiiP. The model suggests a way in which zinc binding to the cytoplasmic fragment creates a docking site to which a metallochaperone can bind for delivery and transport of its zinc cargo. Because the cytoplasmic domain may exist in the cell as an independent, soluble protein, a proposal is advanced that it functions as a metallochaperone and that it regulates the zinc-transporting activity of the full-length protein. The latter requires that zinc binding becomes uncoupled from the creation of a metallochaperone-docking site on CzrB.

Fixed-target serial oscillation crystallography at room temperature.

A fixed-target approach to high-throughput room-temperature serial synchrotron crystallography with oscillation is described. Patterned silicon chips with microwells provide high crystal-loading density with an extremely high hit rate. The microfocus, undulator-fed beamline at CHESS, which has compound refractive optics and a fast-framing detector, was built and optimized for this experiment. The high-throughput oscillation method described here collects 1-5° of data per crystal at room temperature with fast (10°â€…s-1) oscillation rates and translation times, giving a crystal-data collection rate of 2.5 Hz. Partial datasets collected by the oscillation method at a storage-ring source provide more complete data per crystal than still images, dramatically lowering the total number of crystals needed for a complete dataset suitable for structure solution and refinement - up to two orders of magnitude fewer being required. Thus, this method is particularly well suited to instances where crystal quantities are low. It is demonstrated, through comparison of first and last oscillation images of two systems, that dose and the effects of radiation damage can be minimized through fast rotation and low angular sweeps for each crystal.

ADP-ribosyl cyclase; crystal structures reveal a covalent intermediate.

ADP-ribosyl cyclase catalyzes the elimination of nicotinamide from NAD and cyclization to cADPR, a known second messenger in cellular calcium signaling pathways. We have determined to 2.0 A resolution the structure of Aplysia cyclase with ribose-5-phosphate bound covalently at C3' and with the base exchange substrate (BES), pyridylcarbinol, bound to the active site. In addition, further refinement at 2.4 A resolution of the structure of nicotinamide-bound cyclase, which was previously reported, reveals that ribose-5-phosphate is also covalently bound in this structure, and a second nicotinamide site was identified. The structures of native and mutant Glu179Ala cyclase were also solved to 1.7 and 2.0 A respectively. It is proposed that the second nicotinamide site serves to promote cyclization by clearing the active site of the nicotinamide byproduct. Moreover, a ribosylation mechanism can be proposed in which the cyclization reaction proceeds through a covalently bound intermediate.

Improving diffraction resolution using a new dehydration method.

The production of high-quality crystals is one of the major obstacles in determining the three-dimensional structure of macromolecules by X-ray crystallography. It is fairly common that a visually well formed crystal diffracts poorly to a resolution that is too low to be suitable for structure determination. Dehydration has proven to be an effective post-crystallization treatment for improving crystal diffraction quality. Several dehydration methods have been developed, but no single one of them is suitable for all crystals. Here, a new convenient and effective dehydration method is reported that makes use of a dehydrating solution that will not dry out in air for several hours. Using this dehydration method, the resolution of Archaeoglobus fulgidus Cas5a crystals has been increased from 3.2 to 1.95 Šand the resolution of Escherichia coli LptA crystals has been increased from <5 to 3.4 Å.

A visible-light-excited fluorescence method for imaging protein crystals without added dyes.

Fluorescence microscopy methods have seen an increase in popularity in recent years for detecting protein crystals in screening trays. The fluorescence-based crystal detection methods have thus far relied on intrinsic UV-inducible tryptophan fluorescence, nonlinear optics or fluorescence in the visible light range dependent on crystals soaked with fluorescent dyes. In this paper data are presented on a novel visible-light-inducible autofluorescence arising from protein crystals as a result of general stabilization of conjugated double-bond systems and increased charge delocalization due to crystal packing. The visible-light-inducible autofluorescence serves as a complementary method to bright-field microscopy in beamline applications where accurate crystal centering about the rotation axis is essential. Owing to temperature-dependent chromophore stabilization, protein crystals exhibit tenfold higher fluorescence intensity at cryogenic temperatures, making the method ideal for experiments where crystals are cooled to 100 K with a cryostream. In addition to the non-damaging excitation wavelength and low laser power required for imaging, the method can also serve a useful role for differentiating protein crystals from salt crystals in screening trays.

Reduction of lattice disorder in protein crystals by high-pressure cryocooling.

High-pressure cryocooling (HPC) has been developed as a technique for reducing the damage that frequently occurs when macromolecular crystals are cryocooled at ambient pressure. Crystals are typically pressurized at around 200 MPa and then cooled to liquid nitrogen temperature under pressure; this process reduces the need for penetrating cryoprotectants, as well as the damage due to cryocooling, but does not improve the diffraction quality of the as-grown crystals. Here it is reported that HPC using a pressure above 300 MPa can reduce lattice disorder, in the form of high mosaicity and/or nonmerohedral twinning, in crystals of three different proteins, namely human glutaminase C, the GTP pyrophosphokinase YjbM and the uncharacterized protein lpg1496. Pressure lower than 250 MPa does not induce this transformation, even with a prolonged pressurization time. These results indicate that HPC at elevated pressures can be a useful tool for improving crystal packing and hence the quality of the diffraction data collected from pressurized crystals.

Erratum: Room-temperature serial crystallography using a kinetically optimized microfluidic device for protein crystallization and on-chip X-ray diffraction. Corrigendum.

The name of one of the authors in the article by Heymann et al. [(2014), IUCrJ, 1, 349-360] is corrected.[This corrects the article DOI: 10.1107/S2052252514016960.].

Room-temperature serial crystallography using a kinetically optimized microfluidic device for protein crystallization and on-chip X-ray diffraction.

An emulsion-based serial crystallographic technology has been developed, in which nanolitre-sized droplets of protein solution are encapsulated in oil and stabilized by surfactant. Once the first crystal in a drop is nucleated, the small volume generates a negative feedback mechanism that lowers the supersaturation. This mechanism is exploited to produce one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room-temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different unoriented crystals. As proof of concept, the structure of glucose isomerase was solved to 2.1 Å, demonstrating the feasibility of high-throughput serial X-ray crystallography using synchrotron radiation.

A prototype direct-detection CCD for protein crystallography.

The fabrication and testing of a prototype deep-depletion direct-conversion X-ray CCD detector are described. The device is fabricated on 600 µm-thick high-resistivity silicon, with 24 × 24 µm pixels in a 4k × 4k pixel format. Calibration measurements and the results of initial protein crystallography experiments at the Cornell High Energy Synchrotron Source (CHESS) F1 beamline are described, as well as suggested improvements for future versions of the detector.

Inhibition of 5,10-methenyltetrahydrofolate synthetase.

The interaction of 5-formyltetrahydrofolate analogs with murine methenyltetrahydrofolate synthetase (MTHFS) was investigated using steady-state kinetics, molecular modeling, and site-directed mutagenesis. MTHFS catalyzes the irreversible cyclization of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Folate analogs that cannot undergo the rate-limiting step in catalysis were inhibitors of murine MTHFS. 5-Formyltetrahydrohomofolate was an effective inhibitor of murine MTHFS (K(i)=0.7 microM), whereas 5-formyl,10-methyltetrahydrofolate was a weak inhibitor (K(i)=10 microM). The former, but not the latter, was slowly phosphorylated by MTHFS. 5-Formyltetrahydrohomofolate was not a substrate for murine MTHFS, but was metabolized when the MTHFS active site Y151 was mutated to Ala. MTHFS active site residues do not directly facilitate N10 attack on the on the N5-iminium phosphate intermediate, but rather restrict N10 motion around N5. Inhibitors specifically designed to block N10 attack appear to be less effective than the natural 10-formyltetrahydrofolate polyglutamate inhibitors.

Evidence for small ubiquitin-like modifier-dependent nuclear import of the thymidylate biosynthesis pathway.

Perturbations in folate-mediated one-carbon metabolism increase rates of uracil misincorporation into DNA during replication, impair cellular methylation reactions, and increase risk for neural tube defects and cancer. One-carbon metabolism is compromised by folate deficiency and common genetic polymorphisms. In this study, the mechanism for the preferential partitioning of cytoplasmic serine hydroxymethyltransferase (cSHMT)-derived methylenetetrahydrofolate to de novo thymidylate biosynthesis was investigated. The cSHMT enzyme was shown to interact with UBC9 and was a substrate for UBC9-catalyzed small ubiquitin-like modifier (SUMO) modification in vitro. SUMOylated cSHMT was detected in extracts from S phase MCF-7 cells, and cSHMT was shown to localize to the nucleus and nuclear periphery during the S and G(2)/M phases of the cell cycle. A common single nucleotide polymorphism (L474F-cSHMT) impaired the UBC9-cSHMT interaction and inhibited cSHMT SUMOylation in vitro. The three folate-dependent enzymes that constitute the de novo thymidylate biosynthesis pathway, cSHMT, thymidylate synthase, and dihydrofolate reductase, all contain SUMO modification consensus sequences. Compartmentation of the folate-dependent de novo thymidylate biosynthesis pathway in the nucleus accounts for the preferential partitioning of cSHMT-derived folate-activated one-carbon units into thymidylate biosynthesis; the efficiency of nuclear folate metabolism is likely to be modified by the cSHMT L474F polymorphism.

Serinocyclins A and B, cyclic heptapeptides from Metarhizium anisopliae.

Two new cyclic heptapeptides, serinocyclins A (1) and B (2), were isolated from conidia of the entomopathogenic fungus Metarhizium anisopliae. Structures were elucidated by a combination of mass spectrometric, NMR, and X-ray diffraction techniques. Serinocyclin A (1) contains three serine units, a hydroxyproline (Hyp), a beta-alanine (beta-Ala), and two uncommon nonproteinogenic amino acids, 1-aminocyclopropane-1-carboxylic acid (Acc) and gamma-hydroxylysine (HyLys). The peptide sequence established for 1 by NMR is cyclo-(Acc-Hyp-Ser1-HyLys-beta-Ala-Ser2-Ser3). Serinocyclin B (2) has Lys in place of the HyLys unit found in 1. Chiral amino acid analysis indicated the presence in both compounds of one (2 S,4 R)-Hyp, two L-Ser, and one D-Ser residue. A Lys found in the hydrolyzate of 2 was established as D-configured. A crystal structure of 1 established the position of the D-Ser (Ser2) and the absolute configuration of the HyLys unit (2 R,4 S). The absence of methyl groups is unusual among fungal peptides and, along with the charged lysyl side chain and multiple hydroxyl groups, contributes to the polar nature of the compounds. Serinocyclin A produced a sublethal locomotory defect in mosquito larvae at an EC 50 of 59 ppm.

Regulation of de novo purine biosynthesis by methenyltetrahydrofolate synthetase in neuroblastoma.

5-Formyltetrahydrofolate (5-formylTHF) is the only folate derivative that does not serve as a cofactor in folate-dependent one-carbon metabolism. Two metabolic roles have been ascribed to this folate derivative. It has been proposed to 1) serve as a storage form of folate because it is chemically stable and accumulates in seeds and spores and 2) regulate folate-dependent one-carbon metabolism by inhibiting folate-dependent enzymes, specifically targeting folate-dependent de novo purine biosynthesis. Methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme that metabolizes 5-formylTHF and catalyzes its ATP-dependent conversion to 5,10-methenylTHF. This reaction determines intracellular 5-formylTHF concentrations and converts 5-formylTHF into an enzyme cofactor. The regulation and metabolic role of MTHFS in one-carbon metabolism was investigated in vitro and in human neuroblastoma cells. Steady-state kinetic studies revealed that 10-formylTHF, which exists in chemical equilibrium with 5,10-methenylTHF, acts as a tight binding inhibitor of mouse MTHFS. [6R]-10-formylTHF inhibited MTHFS with a K(i) of 150 nM, and [6R,S]-10-formylTHF triglutamate inhibited MTHFS with a K(i) of 30 nm. MTHFS is the first identified 10-formylTHF tight-binding protein. Isotope tracer studies in neuroblastoma demonstrate that MTHFS enhances de novo purine biosynthesis, indicating that MTHFS-bound 10-formylTHF facilitates de novo purine biosynthesis. Feedback metabolic regulation of MTHFS by 10-formylTHF indicates that 5-formylTHF can only accumulate in the presence of 10-formylTHF, providing the first evidence that 5-formylTHF is a storage form of excess formylated folates in mammalian cells. The sequestration of 10-formylTHF by MTHFS may explain why de novo purine biosynthesis is protected from common disruptions in the folate-dependent one-carbon network.

Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folate-independent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme. The cleavage of 3-hydroxy amino acids to glycine and an aldehyde occurs by a retroaldol mechanism. However, the folate-dependent cleavage of serine can be described by either the same retroaldol mechanism with formaldehyde as an enzyme-bound intermediate or by a nucleophilic displacement mechanism in which N5 of tetrahydrofolate displaces the C3 hydroxyl of serine, forming a covalent intermediate. Glu75 of SHMT is clearly involved in the reaction mechanism; it is within hydrogen bonding distance of the hydroxyl group of serine and the formyl group of 5-formyltetrahydrofolate in complexes of these species with SHMT. This residue was changed to Leu and Gln, and the structures, kinetics, and spectral properties of the site mutants were determined. Neither mutation significantly changed the structure of SHMT, the spectral properties of its complexes, or the kinetics of the retroaldol cleavage of allothreonine and 3-phenylserine. However, both mutations blocked the folate-dependent serine-to-glycine reaction and the conversion of methenyltetrahydrofolate to 5-formyltetrahydrofolate. These results clearly indicate that interaction of Glu75 with folate is required for folate-dependent reactions catalyzed by SHMT. Moreover, we can now propose a promising modification to the retroaldol mechanism for serine cleavage. As the first step, N5 of tetrahydrofolate makes a nucleophilic attack on C3 of serine, breaking the C2-C3 bond to form N5-hydroxymethylenetetrahydrofolate and an enzyme-bound glycine anion. The transient formation of formaldehyde as an intermediate is possible, but not required. This mechanism explains the greatly enhanced rate of serine cleavage in the presence of folate, and avoids some serious difficulties presented by the nucleophilic displacement mechanism involving breakage of the C3-OH bond.