Solution NMR Determination of the CDHR3 Rhinovirus-C Binding Domain, EC1.

Cadherin Related Family Member 3 (CDHR3) is the identified and required cellular receptor for all virus isolates in the rhinovirus-C species (RV-C). Cryo-EM determinations recently resolved the atomic structure of RV-C15a, and subsequently, a complex of this virus bound to CDHR3 extracellular domain 1 (EC1), the N-terminal portion of this receptor responsible for virus interactions. The EC1 binds to a hypervariable sequence footprint on the virus surface, near the 3-fold axis of icosahedral symmetry.

ISCU interacts with NFU1, and ISCU[4Fe-4S] transfers its Fe-S cluster to NFU1 leading to the production of holo-NFU1.

NFU1 is a late-acting factor in the biogenesis of human mitochondrial iron-sulfur proteins. Mutations in NFU1 are associated with genetic diseases such as multiple mitochondrial dysfunctions syndrome 1 (MMDS1) that involve defects in mitochondrial [4Fe-4S] proteins. We present results from NMR spectroscopy, small angle X-ray scattering, size exclusion chromatography, and isothermal titration calorimetry showing that the structured conformer of human ISCU binds human NFU1.

Function and solution structure of the Arabidopsis thaliana RALF8 peptide.

We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine-rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well-resolved nuclear magnetic resonance (NMR) spectra and also biological activity through interaction with a plant receptor kinase, cytoplasmic calcium mobilization, and in vivo root growth suppression. By contrast, RALF1 could only be isolated from inclusion bodies as a construct containing an N-terminal His-tag; its poorly resolved NMR spectrum was indicative of aggregation.

Architectural Features of Human Mitochondrial Cysteine Desulfurase Complexes from Crosslinking Mass Spectrometry and Small-Angle X-Ray Scattering.

Cysteine desulfurase plays a central role in mitochondrial iron-sulfur cluster biogenesis by generating sulfur through the conversion of L-cysteine to L-alanine and by serving as the platform for assembling other components of the biosynthetic machinery, including ISCU, frataxin, and ferredoxin. The human mitochondrial cysteine desulfurase complex consists of two copies each of NFS1, ISD11, and acyl carrier protein. We describe results from chemical crosslinking coupled with tandem mass spectrometry and small-angle X-ray scattering studies that are consistent with a closed NFS1 dimer rather than an open one for both the cysteine desulfurase-ISCU and cysteine desulfurase-ISCU-frataxin complexes.

Interactions of iron-bound frataxin with ISCU and ferredoxin on the cysteine desulfurase complex leading to Fe-S cluster assembly.

Frataxin (FXN) is involved in mitochondrial iron‑sulfur (Fe-S) cluster biogenesis and serves to accelerate Fe-S cluster formation. FXN deficiency is associated with Friedreich ataxia, a neurodegenerative disease. We have used a combination of isothermal titration calorimetry and multinuclear NMR spectroscopy to investigate interactions among the components of the biological machine that carries out the assembly of iron‑sulfur clusters in human mitochondria.

ISCU(M108I) and ISCU(D39V) Differ from Wild-Type ISCU in Their Failure To Form Cysteine Desulfurase Complexes Containing Both Frataxin and Ferredoxin.

Whereas iron-sulfur (Fe-S) cluster assembly on the wild-type scaffold protein ISCU, as catalyzed by the human cysteine desulfurase complex (NIA), exhibits a requirement for frataxin (FXN), in yeast, ISCU variant M108I has been shown to bypass the FXN requirement. Wild-type ISCU populates two interconverting conformational states: one structured and one dynamically disordered. We show here that variants ISCU(M108I) and ISCU(D39V) of human ISCU populate only the structured state.

Mitochondrial Cysteine Desulfurase and ISD11 Coexpressed in Escherichia coli Yield Complex Containing Acyl Carrier Protein.

Mitochondrial cysteine desulfurase is an essential component of the machinery for iron-sulfur cluster biosynthesis. It has been known that human cysteine desulfurase that is catalytically active in vitro can be prepared by overexpressing in Escherichia coli cells two protein components of this system, the cysteine desulfurase protein NFS1 and the auxiliary protein ISD11. We report here that this active preparation contains, in addition, the holo-form of E.

Human Mitochondrial Ferredoxin 1 (FDX1) and Ferredoxin 2 (FDX2) Both Bind Cysteine Desulfurase and Donate Electrons for Iron-Sulfur Cluster Biosynthesis.

Ferredoxins play an important role as an electron donor in iron-sulfur (Fe-S) cluster biosynthesis. Two ferredoxins, human mitochondrial ferredoxin 1 (FDX1) and human mitochondrial ferredoxin 2 (FDX2), are present in the matrix of human mitochondria. Conflicting results have been reported regarding their respective function in mitochondrial iron-sulfur cluster biogenesis.

Structural/Functional Properties of Human NFU1, an Intermediate [4Fe-4S] Carrier in Human Mitochondrial Iron-Sulfur Cluster Biogenesis.

Human mitochondrial NFU1 functions in the maturation of iron-sulfur proteins, and NFU1 deficiency is associated with a fatal mitochondrial disease. We determined three-dimensional structures of the N- and C-terminal domains of human NFU1 by nuclear magnetic resonance spectroscopy and used these structures along with small-angle X-ray scattering (SAXS) data to derive structural models for full-length monomeric apo-NFU1, dimeric apo-NFU1 (an artifact of intermolecular disulfide bond formation), and holo-NFUI (the [4Fe-4S] cluster-containing form of the protein). Apo-NFU1 contains two cysteine residues in its C-terminal domain, and two apo-NFU1 subunits coordinate one [4Fe-4S] cluster to form a cluster-linked dimer.

The Complex Energy Landscape of the Protein IscU.

IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, traverses a complex energy landscape during Fe-S cluster synthesis and transfer. Our previous studies showed that IscU populates two interconverting conformational states: one structured (S) and one largely disordered (D). Both states appear to be functionally important because proteins involved in the assembly or transfer of Fe-S clusters have been shown to interact preferentially with either the S or D state of IscU.

Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies.

Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or (1)H-(15)N HSQC NMR analyses.

The specialized Hsp70 (HscA) interdomain linker binds to its nucleotide-binding domain and stimulates ATP hydrolysis in both cis and trans configurations.

Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron-sulfur (Fe-S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min(-1) at 25 °C, henceforth reported in units of min(-1)).

Nucleotide-dependent interactions within a specialized Hsp70/Hsp40 complex involved in Fe-S cluster biogenesis.

The structural mechanism by which Hsp70-type chaperones interact with Hsp40-type co-chaperones has been of great interest, yet still remains a matter of debate. Here, we used solution NMR spectroscopy to investigate the ATP-/ADP-dependent interactions between Escherichia coli HscA and HscB, the specialized Hsp70/Hsp40 molecular chaperones that mediate iron-sulfur cluster transfer. We observed that NMR signals assigned to amino acid residues in the J-domain and its "HPD" motif of HscB broadened severely upon the addition of ATP-bound HscA, but these signals were not similarly broadened by ADP-bound HscA or the isolated nucleotide binding domain of HscA complexed with either ATP or ADP.

Solution structure of the 2A protease from a common cold agent, human rhinovirus C2, strain W12.

Human rhinovirus strains differ greatly in their virulence, and this has been correlated with the differing substrate specificity of the respective 2A protease (2Apro). Rhinoviruses use their 2Apro to cleave a spectrum of cellular proteins important to virus replication and anti-host activities. These enzymes share a chymotrypsin-like fold stabilized by a tetra-coordinated zinc ion.

Role of IscX in iron-sulfur cluster biogenesis in Escherichia coli.

The Escherichia coli isc operon encodes key proteins involved in the biosynthesis of iron-sulfur (Fe-S) clusters. Whereas extensive studies of most ISC proteins have revealed their functional properties, the role of IscX (also dubbed YfhJ), a small acidic protein encoded by the last gene in the operon, has remained in question. Previous studies showed that IscX binds iron ions and interacts with the cysteine desulfurase (IscS) and the scaffold protein for cluster assembly (IscU), and it has been proposed that IscX functions either as an iron supplier or a regulator of Fe-S cluster biogenesis.

Human mitochondrial chaperone (mtHSP70) and cysteine desulfurase (NFS1) bind preferentially to the disordered conformation, whereas co-chaperone (HSC20) binds to the structured conformation of the iron-sulfur cluster scaffold protein (ISCU).

Human ISCU is the scaffold protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis and transfer. NMR spectra have revealed that ISCU populates two conformational states; that is, a more structured state (S) and a partially disordered state (D). We identified two single amino acid substitutions (D39V and N90A) that stabilize the S-state and two (D39A and H105A) that stabilize the D-state.

[2Fe-2S]-ferredoxin binds directly to cysteine desulfurase and supplies an electron for iron-sulfur cluster assembly but is displaced by the scaffold protein or bacterial frataxin.

Escherichia coli [2Fe-2S]-ferredoxin (Fdx) is encoded by the isc operon along with other proteins involved in the 'house-keeping' mechanism of iron-sulfur cluster biogenesis. Although it has been proposed that Fdx supplies electrons to reduce sulfane sulfur (S(0)) produced by the cysteine desulfurase (IscS) to sulfide (S(2-)) as required for the assembly of Fe-S clusters on the scaffold protein (IscU), direct experimental evidence for the role of Fdx has been lacking. Here, we show that Fdx (in either oxidation state) interacts directly with IscS.

Specialized Hsp70 chaperone (HscA) binds preferentially to the disordered form, whereas J-protein (HscB) binds preferentially to the structured form of the iron-sulfur cluster scaffold protein (IscU).

The Escherichia coli protein IscU serves as the scaffold for Fe-S cluster assembly and the vehicle for Fe-S cluster transfer to acceptor proteins, such as apoferredoxin. IscU populates two conformational states in solution, a structured conformation (S) that resembles the conformation of the holoprotein IscU-[2Fe-2S] and a dynamically disordered conformation (D) that does not bind metal ions. NMR spectroscopic results presented here show that the specialized Hsp70 chaperone (HscA), alone or as the HscA-ADP complex, preferentially binds to and stabilizes the D-state of IscU.

Structural characterization of Hsp12, the heat shock protein from Saccharomyces cerevisiae, in aqueous solution where it is intrinsically disordered and in detergent micelles where it is locally α-helical.

Hsp12 (heat shock protein 12) belongs to the small heat shock protein family, partially characterized as a stress response, stationary phase entry, late embryonic abundant-like protein located at the plasma membrane to protect membrane from desiccation. Here, we report the structural characterization of Hsp12 by NMR and biophysical techniques. The protein was labeled uniformly with nitrogen-15 and carbon-13 so that its conformation could be determined in detail both in aqueous solution and in two membrane-mimetic environments, SDS and dodecylphosphocholine (DPC) micelles.

Robotic large-scale application of wheat cell-free translation to structural studies including membrane proteins.

The use of the Protemist XE, an automated discontinuous-batch protein synthesis robot, in cell-free translation is reported. The soluble Galdieria sulphuraria protein DCN1 was obtained in greater than 2mg total synthesis yield per mL of reaction mixture from the Protemist XE, and the structure was subsequently solved by X-ray crystallography using material from one 10 mL synthesis (PDB ID: 3KEV). The Protemist XE was also capable of membrane protein translation.

Rapid, robotic, small-scale protein production for NMR screening and structure determination.

Three-dimensional protein structure determination is a costly process due in part to the low success rate within groups of potential targets. Conventional validation methods eliminate the vast majority of proteins from further consideration through a time-consuming succession of screens for expression, solubility, purification, and folding. False negatives at each stage incur unwarranted reductions in the overall success rate.

X-ray structure of Danio rerio secretagogin: A hexa-EF-hand calcium sensor.

Many essential physiological processes are regulated by the modulation of calcium concentration in the cell. The EF-hand proteins represent a superfamily of calcium-binding proteins involved in calcium signaling and homeostasis. Secretagogin is a hexa-EF-hand protein that is highly expressed in pancreatic islet of Langerhans and neuroendocrine cells and may play a role in the trafficking of secretory granules.

The Center for Eukaryotic Structural Genomics.

The Center for Eukaryotic Structural Genomics (CESG) is a "specialized" or "technology development" center supported by the Protein Structure Initiative (PSI). CESG's mission is to develop improved methods for the high-throughput solution of structures from eukaryotic proteins, with a very strong weighting toward human proteins of biomedical relevance. During the first three years of PSI-2, CESG selected targets representing 601 proteins from Homo sapiens, 33 from mouse, 10 from rat, 139 from Galdieria sulphuraria, 35 from Arabidopsis thaliana, 96 from Cyanidioschyzon merolae, 80 from Plasmodium falciparum, 24 from yeast, and about 25 from other eukaryotes.

Structural and functional characterization of a novel phosphatase from the Arabidopsis thaliana gene locus At1g05000.

The crystal structure of the protein product of the gene locus At1g05000, a hypothetical protein from A. thaliana, was determined by the multiple-wavelength anomalous diffraction method and was refined to an R factor of 20.4% (R(free) = 24.