Natalia Oganesyan

Identification of the Protein C/Activated Protein C Binding Sites on the Endothelial Cell Protein C Receptor IMPLICATIONS FOR A NOVEL MODE OF LIGAND RECOGNITION BY A MAJOR HISTOCOMPATIBILITY COMPLEX CLASS 1-TYPE RECEPTOR

The endothelial cell protein C receptor (EPCR) is an endothelial cell-specific transmembrane protein that binds both protein C and activated protein C (APC). EPCR regulates the protein C anticoagulant pathway by binding protein C and augmenting protein C activation by the thrombin-thrombomodulin complex. EPCR is homologous to the MHC class 1/CD1 family, members of which contain two α-helices that sit upon an 8-stranded β-sheet platform. In this study, we identified 10 residues that, when mutated to alanine, result in the loss of protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87, Phe-146, Tyr-154, Thr-157, Arg-158, and Glu-160). Glutamine substitutions at the four N-linked carbohydrate attachment sites of EPCR have little affect on APC binding, suggesting that the carbohydrate moieties of EPCR are not critical for ligand recognition. We then mapped the epitopes for four anti-human EPCR monoclonal antibodies (mAbs), two of which block EPCR/Fl-APC (APC labeled at the active site with fluorescein) interactions, whereas two do not. These epitopes were localized by generating human-mouse EPCR chimeric proteins, since the mAbs under investigation do not recognize mouse EPCR. We found that 5 of the 10 candidate residues for protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87) colocalize with the epitope for one of the blocking mAbs. Three-dimensional molecular modeling of EPCR indicates that the 10 protein C/APC binding candidate residues are clustered at the distal end of the two α-helical segments. Protein C activation studies on 293 cells that coexpress EPCR variants and thrombomodulin demonstrate that protein C binding to EPCR is necessary for the EPCR-dependent enhancement in protein activation by the thrombin-thrombomodulin complex. These studies indicate that EPCR has exploited the MHC class 1 fold for an alternative and possibly novel mode of ligand recognition. These studies are also the first to identify the protein C/APC binding region of EPCR and may provide useful information about molecular defects in EPCR that could contribute to cardiovascular disease susceptibility.

Structural characterization of an iron-sulfur cluster assembly protein IscU in a zinc-bound form.

Introduction. Iron–sulfur (Fe-S) clusters are simple inorganic prosthetic groups widely distributed in nature. Proteins that contain Fe-S clusters play essential roles in diverse biological processes including electron transfer, gene regulation, environmental sensing, and substrate activation. Although it is possible to assemble Fe-S clusters into proteins from inorganic sulfide and iron in vitro, the biogenesis of Fe-S cluster in vivo, however, appears to be facilitated by proteins rather than spontaneous formation. Recent studies have led to the discovery that a highly conserved gene cluster iscSUA-hscBA-fdx is essential for the biogenesis of Fe-S cluster proteins in bacteria. Proteins encoded by this gene cluster include: IscS, IscU, IscA, HscA, HscB, and Ferredoxin (Fd). Homologies of these proteins have also been identified in eukaryotic organisms, indicating a conserved mechanism for the biogenesis of Fe-S proteins. Further biochemical studies have revealed detailed roles of these Fe-S cluster proteins. IscS, a homolog of NifS, is a homodimeric pyridoxal phosphate-dependent cysteine desulfurase. It catalyzes the desulfurization of L-cysteine to L-alanine and provides S to IscU for Fe-S cluster assembly. IscU provides a scaffold for the assembly of a nascent iron– sulfur cluster prior to its delivery to apo Fe-S proteins. IscA is proposed to function as an alternative scaffold for Fe-S cluster assembly, but the exact role of this protein is still not determined. HscA and HscB are molecular chaperones that can selectively bind IscU and assist in the biogenesis of Fe-S proteins. The detailed roles of these molecular chaperones are also not clear. Sequence comparisons suggest IscU is a highly conserved protein and is homologous to the N-terminal domain of NifU (nNifU), an essential protein for nitrogen fixation. IscU/nNifU contains three strictly conserved cysteine residues. Site-directed mutagenesis data suggest that all three cysteine residues are essential for the function of IscU/nNifU proteins. Biochemical assay showed IscS can form a covalent complex with IscU through residue Cys328 in IscS and residue Cys63 in IscU in Escherichia coli. Characterization of Fe-S cluster assembly on IscU or ISU (eukaryotic IscU) from several organisms including Azotobacter vinelandii, E. coli, Thermotoga maritima (Tm) and human have been carried out. In presence of IscS/NifS, D-cysteine, Fe and reducing agent, IscU/nNifU is able to assemble a transient [Fe2S2] 2

An automated small-scale protein expression and purification screening provides beneficial information for protein production

One of the first key steps in structural genomics is high-throughput expression and rapid screening to select highly soluble proteins, the preferred candidates for crystal production. Here we describe the methodology used at the Berkeley Structural Genomics Center (BSGC) for automated parallel expression and small-scale purification of fusion proteins using a 96-well format. Our robotic method includes cell lysis, soluble fraction separation and purification with affinity resins. For detection of His-tagged proteins in the soluble fractions and after affinity resin elution, a dot-blot procedure with an anti-His-antibody is used. The expression level and molecular mass of recombinant proteins are checked by SDS-PAGE. With this approach, we are able to obtain beneficial information to be used for large-scale protein expression and purification.

On-column protein refolding for crystallization.

One major bottleneck in protein production in Escherichia coli for structural genomics projects is the formation of insoluble protein aggregates (inclusion bodies). The efficient refolding of proteins from inclusion bodies is becoming an important tool that can provide soluble native proteins for structural and functional studies. Here we report an on-column refolding method established at the Berkeley Structural Genomics Center (BSGC). Our method is a combination of an ‘artificial chaperone-assisted refolding’ method previously proposed and affinity chromatography to take advantage of a chromatographic step: less time-consuming, no filtration or concentration, with the additional benefit of protein purification. It can be easily automated and formatted for high-throughput process.

Crystal Structure of the “PhoU-Like” Phosphate Uptake Regulator from Aquifex aeolicus

The phoU gene of Aquifex aeolicus encodes a protein called PHOU_AQUAE with sequence similarity to the PhoU protein of Escherichia coli. Despite the fact that there is a large number of family members (more than 300) attributed to almost all known bacteria and despite PHOU_AQUAEs association with the regulation of genes for phosphate metabolism, the nature of its regulatory function is not well understood. Nearly one-half of these PhoU-like proteins, including both PHOU_AQUAE and the one from E. coli, form a subfamily with an apparent dimer structure of two PhoU domains on the basis of their amino acid sequence. The crystal structure of PHOU_AQUAE (a 221-amino-acid protein) reveals two similar coiled-coil PhoU domains, each forming a three-helix bundle. The structures of PHOU_AQUAE proteins from both a soluble fraction and refolded inclusion bodies (at resolutions of 2.8 and 3.2A, respectively) showed no significant differences. The folds of the PhoU domain and Bag domains (for a class of cofactors of the eukaryotic chaperone Hsp70 family) are similar. Accordingly, we propose that gene regulation by PhoU may occur by association of PHOU_AQUAE with the ATPase domain of the histidine kinase PhoR, promoting release of its substrate PhoB. Other proteins that share the PhoU domain fold include the coiled-coil domains of the STAT protein, the ribosome-recycling factor, and structural proteins like spectrin.