Galina Obmolova

Crystal structure of the Escherichia coli YcdX protein reveals a trinuclear zinc active site

Alexey Teplyakov, Galina Obmolova, Pavel P. Khil, Andrew J. Howard, R. Daniel Camerini-Otero, and Gary L. Gilliland Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute and the National Institute of Standards and Technology, Rockville, Maryland Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland Center for Synchrotron Radiation Research and Instrumentation, Biological, Chemical and Physical Sciences Department, Illinois Institute of Technology, Chicago, Illinois

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody-antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1-20mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS-PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.

Crystal Structure of the YchF Protein Reveals Binding Sites for GTP and Nucleic Acid

The bacterial protein encoded by the gene ychF is 1 of 11 universally conserved GTPases and the only one whose function is unknown. The crystal structure determination of YchF was sought to help with the functional assignment of the protein. The YchF protein from Haemophilus influenzae was cloned and expressed, and the crystal structure was determined at 2.4 A resolution. The polypeptide chain is folded into three domains. The N-terminal domain has a mononucleotide binding fold typical for the P-loop NTPases. An 80-residue domain next to it has a pronounced alpha-helical coiled coil. The C-terminal domain features a six-stranded half-barrel that curves around an alpha-helix. The crablike three-domain structure of YchF suggests the binding site for a double-stranded nucleic acid in the cleft between the domains. The structure of the putative GTP-binding site is consistent with the postulated guanine specificity of the protein. Fluorescence measurements have demonstrated the ability of YchF to bind a double-stranded nucleic acid and GTP. Taken together with other experimental data and genomic analysis, these results suggest that YchF may be part of a nucleoprotein complex and may function as a GTP-dependent translation factor.

Crystal structure of the YjeE protein from Haemophilus influenzae: a putative Atpase involved in cell wall synthesis

A hypothetical protein encoded by the gene YjeE of Haemophilus influenzae was selected as part of a structural genomics project for X‐ray analysis to assist with the functional assignment. The protein is considered essential to bacteria because the gene is present in virtually all bacterial genomes but not in those of archaea or eukaryotes. The amino acid sequence shows no homology to other proteins except for the presence of the Walker A motif G‐X‐X‐X‐X‐G‐K‐T that indicates the possibility of a nucleotide‐binding protein. The YjeE protein was cloned, expressed, and the crystal structure determined by the MAD method at 1.7‐Å resolution. The protein has a nucleotide‐binding fold with a four‐stranded parallel β‐sheet flanked by antiparallel β‐strands on each side. The topology of the β‐sheet is unique among P‐loop proteins and has features of different families of enzymes. Crystallization of YjeE in the presence of ATP and Mg2+ resulted in the structure with ADP bound in the P‐loop. The ATPase activity of YjeE was confirmed by kinetic measurements. The distribution of conserved residues suggests that the protein may work as a “molecular switch” triggered by ATP hydrolysis. The phylogenetic pattern of YjeE suggests its involvement in cell wall biosynthesis. Proteins 2002;48:220–226.

Crystal structure of Escherichia coli protein ybgI, a toroidal structure with a dinuclear metal site

BackgroundThe protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.ResultsThe structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.ConclusionThe toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.

Crystal Structure of the YgfZ Protein from Escherichia coli Suggests a Folate-Dependent Regulatory Role in One-Carbon Metabolism

The ygfZ gene product of Escherichia coli represents a large protein family conserved in bacteria to eukaryotes. The members of this family are uncharacterized proteins with marginal sequence similarity to the T-protein (aminomethyltransferase) of the glycine cleavage system. To assist with the functional assignment of the YgfZ family, the crystal structure of the E. coli protein was determined by multiwavelength anomalous diffraction. The protein molecule has a three-domain architecture with a central hydrophobic channel. The structure is very similar to that of bacterial dimethylglycine oxidase, an enzyme of the glycine betaine pathway and a homolog of the T-protein. Based on structural superposition, a folate-binding site was identified in the central channel of YgfZ, and the ability of YgfZ to bind folate derivatives was confirmed experimentally. However, in contrast to dimethylglycine oxidase and T-protein, the YgfZ family lacks amino acid conservation at the folate site, which implies that YgfZ is not an aminomethyltransferase but is likely a folate-dependent regulatory protein involved in one-carbon metabolism.

Crystal structure of the bacterial YhcH protein indicates a role in sialic acid catabolism.

The yhcH gene is part of the nan operon in bacteria that encodes proteins involved in sialic acid catabolism. Determination of the crystal structure of YhcH from Haemophilus influenzae was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. The structure was determined at 2.2-A resolution by multiple-wavelength anomalous diffraction. The protein fold is a variation of the double-stranded beta-helix. Two antiparallel beta-sheets form a funnel opened at one side, where a putative active site contains a copper ion coordinated to the side chains of two histidine and two carboxylic acid residues. A comparison to other proteins with a similar fold and analysis of the genomic context suggested that YhcH may be a sugar isomerase involved in processing of exogenous sialic acid.

Structure of the EMMPRIN N-terminal domain 1: dimerization via beta-strand swapping.

Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as Hab18G, CD147, Basigin, M6, and neurothelin, is a membrane glycoprotein expressed on the surface of various cell types and many cancer cells. EMMPRIN stimulates adjacent fibroblasts and tumor cells to produce matrix metalloproteinases and plays an important role in tumor invasion and metastasis, angiogenesis, spermatogensis and fertilization, cell-cell adhesion and communication, and other biological processes (reviewed in Ref. 1 and references therein). It was demonstrated that the EMMPRIN extracellular domain (ECD), which structurally belongs to the IgG superfamily, can form homo-oligomers in a cis dependent manner and the N-terminal domain 1 (residues 22-101) was necessary and sufficient to mediate this interaction. The crystal structure of the ECD of recombinant human EMMPRIN (Hab18G/CD147) expressed in E. coli was reported at 2.8 {angstrom} resolution (Yu et al. 2008). The construct consists of residues 22-205 of the mature protein and has both an N-terminal IgC2 domain (ND1, residues 22-101) and a C-terminal IgC2 domain (ND2, residues 107-205). The two domains are joined by a five amino acid residue linker that constitutes a flexible hinge between the two domains. The crystal form has four copies of the molecule in the asymmetric unit, each of which hasmorexa0» a different inter-domain angle that varies from 121{sup o} to 144{sup o}. The two domains each have a conserved disulfide bridge and both are comprised of two {beta}-sheets formed by strands EBA and GFCC, and DEBA and AGFCC for ND1 and ND2, respectively. Based on the crystal packing in this structure, the authors proposed that lateral packing between the two IgG domains of EMMPRIN ECD represents a potential mechanism for cell adhesion. Here we report the 2.0-{angstrom} crystal structure of the N-terminal domain of EMMPRIN ECD (ND1) expressed in mammalian cells. The overall structure of the domain is very similar to that in the full length ECD. Quite unexpectedly, ND1 forms a dimer mediated through the exchange of its last {beta}-strand (strand G). {beta}-strand swapping, which is a subset of 3D domain swapping, has been found to mediate cell-cell adhesion by cadherins. 3D domain swapping has been proposed to be a mechanism of protein oligomerization, aggregation, evolution of oligomeric proteins from single domains and amyloidogenesis. In domain swapped proteins, the same structural elements are involved in the final 3D structure, and so there is little overall energetic difference between the monomer and the swapped oligomers. However, there is often a high energy barrier for the conversion as it often goes through an unfolded state. It is also possible that strand-swapping occurs during folding of nascent polypeptide chains. Frequently, the exchange hinges contain proline-rich motifs which are often in high strain conformations. Domain swapping appears to be a strategy to resolve such local structural strain. The exchange hinge of ND1 contains a Pro-Glu-Pro tripeptide motif. Both of the proline residues adopt extended trans conformations, when compared with cis in the full-length ECD structure. Proline cis-trans isomerization may be the driving force for this exchange. Strand-exchanged dimerization may be a mechanism for the oligomerization of EMMPRIN ECD and its cis-dependent homophilic interactions in cell-cell adhesion.«xa0less

Crystal structure of D-serine dehydratase from Escherichia coli.

D-Serine dehydratase from Escherichia coli is a member of the β-family (fold-type II) of the pyridoxal 5-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97Å-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55Å resolution. The structure of DSD reveals a larger pyridoxal 5-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the β-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279-Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.

Crystal structure of the Escherichia coli YjiA protein suggests a GTP-dependent regulatory function†

Pavel P. Khil, Galina Obmolova, Alexey Teplyakov, Andrew J. Howard, Gary L. Gilliland, and R. Daniel Camerini-Otero Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute and the National Institute of Standards and Technology, Rockville, Maryland Center for Synchrotron Radiation Research and Instrumentation, Biological, Chemical and Physical Sciences Department, Illinois Institute of Technology, Chicago, Illinois