Geng Meng

Structural and functional characterization of K339T substitution identified in the PB2 subunit cap-binding pocket of influenza A virus

Background: Amino acid changes in PB2 are associated with evolution of influenza virus. Results: K339T substitution in PB2cap reduces the cap binding affinity, polymerase activity, RNA synthesis activity, and murine mortality. Conclusion: Substitution in PB2cap modulates the polymerase activity and virulence by regulating the cap binding activity. Significance: We identified and characterized an emerging K339T substitution in PB2cap. Influenza virus RNA-dependent RNA polymerase is a heterotrimer composed of PA, PB1, and PB2 subunits. RNA-dependent RNA polymerase is required for both transcription and replication of influenza viral RNA taking place in the nucleus of infected cells. A “cap-snatching” mechanism is used to generate a 5′-capped primer for transcription in which the cap-binding domain of PB2 (PB2cap) captures the 5′ cap of the host pre-mRNA. Our statistical analysis of PB2 sequences showed that residue Lys339 located in the cap-binding pocket of H5N1 PB2cap was gradually replaced by Thr339 over the past decade. To understand the role of this amino acid polymorphism, we solved the crystal structures of PB2cap with or without a pre-mRNA cap analog, m7GTP, in the presence of Lys339 or Thr339. The structures showed that Lys339 contributes to binding the γ-phosphate group of m7GTP, and the replacement of Lys339 by Thr eliminates this interaction. Isothermal titration calorimetry analysis showed that Thr339 attenuated the PB2cap cap binding activity in vitro compared with Lys339. Further functional studies confirmed that Thr339-PB2-containing ribonucleoprotein complex has a reduced influenza polymerase activity and RNA synthesis activity, and a reconstituted H5N1 virus containing the Thr339 substitution exhibited a lower virulence to mice but more active replication in Madin-Darby canine kidney cells. The K339T substitution in the cap-binding pocket of PB2 modulates the polymerase activity and virulence by regulating the cap binding activity. It is informative to track variations in the cap-binding pocket of PB2 in surveillance of the evolution and spread of influenza virus.

Structure of Human Stabilin-1 Interacting Chitinase-like Protein (SI-CLP) Reveals a Saccharide-binding Cleft with Lower Sugar-binding Selectivity

Human secreted protein stabilin-1 interacting chitinase-like protein (SI-CLP) has been identified as a novel member of Glyco_18 domain-containing proteins that is involved in host defense and inflammatory reactions. Efficient secretion of SI-CLP is mediated by its interaction with the endocytic/sorting receptor stabilin-1. SI-CLP is expressed abundantly in macrophages and neutrophils and is up-regulated by Th2 cytokine IL-4 and glucocorticoid, which suggest that SI-CLP could be a marker for adverse effects of glucocorticoid therapy. To gain insight into the biological function of SI-CLP, we determined the crystal structure of SI-CLP at 2.7 Å resolution by x-ray crystallography and found that it featured a typical triose-phosphate isomerase barrel fold with a putative saccharide-binding cleft. Comparison with other chitinase-like proteins showed the cleft to be atypically wide and open. The saccharide-binding capacity of SI-CLP was investigated, and its ligand-binding specificity was found to relate to the length of the oligosaccharides, with preference for chitotetraose. Further investigations reveal that SI-CLP could bind LPS in vitro and neutralize its endotoxin effect on macrophages. Our results demonstrate the saccharide-binding property of SI-CLP by structure and in vitro biochemical analyses and suggest the possible roles of SI-CLP in pathogen sensing and endotoxin neutralization.

A novel nuclear‐localized protein with special adenylate kinase properties from Caenorhabditis elegans

The adrenal gland protein AD‐004 like protein (ADLP) from Caenorhabditis elegans was cloned and expressed in Escherichia coli. Enzyme assays showed that ADLP has special adenylate kinase (AK) properties, with ATP and dATP as the preferred phosphate donors. In contrast to all other AK isoforms, AMP and dAMP were the preferred substrates of ADLP; CMP, TMP and shikimate acid were also good substrates. Subcellular localization studies showed a predominant nuclear localization for this protein, which is different from AK1–AK5, but similar to that of human AK6. These results suggest that ADLP is more likely a member of the AK6 family. Furthermore, RNAi experiments targeting ADLP were conducted and showed that RNAi treatment resulted in the suppression of worm growth.

NADPH Is an Allosteric Regulator of HSCARG

NADP(H) is an important cofactor that controls many fundamental cellular processes. We have determined the crystal structure of HSCARG, a novel NADPH sensor, and found that it forms an asymmetrical dimer with only one subunit occupied by an NADPH molecule, and the two subunits have dramatically different conformations. To study the role of NADPH in affecting the structure and function of HSCARG, here, we constructed a series of HSCARG mutants to abolish NADPH binding ability. Protein structures of two mutants, R37A and Y81A, were solved by X-ray crystallography. The dimerization of wild-type and mutant HSCARG was studied by dynamic light scattering. Differences between the function of wild-type and mutant HSCARG were also compared. Our results show that binding of NADPH is necessary for HSCARG to form a stable asymmetric dimer. The conformation of the monomeric mutants was similar to that of NADPH-bound Molecule I in wild-type HSCARG, although some conformational changes were found in the NADPH binding site. Furthermore, we also noticed that abolition of NADPH binding ability changes the distribution of HSCARG in the cell and that these mutants without NADPH are more strongly associated with argininosuccinate synthetase as compared with wild-type HSCARG. These data suggest that NADPH functions as an allosteric regulator of the structure and function of HSCARG. In response to the changes in the NADPH/NADP(+) ratio within cells, HSCARG, as a redox sensor, associates and dissociates with NADPH to form a new dynamic equilibrium. This equilibrium, in turn, will tip the dimerization balance of the protein molecule and consequently controls the regulatory function of HSCARG.

Rigidity of Wedge Loop in PACSIN 3 Protein Is a Key Factor in Dictating Diameters of Tubules

Background: PACSINs participate in cellular membrane remodeling. Results: PACSIN 3 F-BAR induces different tubules compared with PACSIN 1 and 2. Structures of PACSINs reveal a novel wedge loop-mediated lateral interaction and different packing mode in PACSIN 3. Conclusion: The rigidity of the wedge loop determines the angles between neighboring dimers and further dictates tubule diameters. Significance: The study provides novel insights into F-BAR domain-induced membrane deformation. BAR (Bin/amphiphysin/Rvs) domain-containing proteins participate in cellular membrane remodeling. The F-BAR proteins normally generate low curvature tubules. However, in the PACSIN subfamily, the F-BAR domain from PACSIN 1 and 2 can induce both high and low curvature tubules. We found that unlike PACSIN 1 and 2, PACSIN 3 could only induce low curvature tubules. To elucidate the key factors that dictate the tubule curvature, crystal structures of all three PACSIN F-BAR domains were determined. A novel type of lateral interaction mediated by a wedge loop is observed between the F-BAR neighboring dimers. Comparisons of the structures of PACSIN 3 with PACSIN 1 and 2 indicate that the wedge loop of PACSIN 3 is more rigid, which influences the lateral interactions between assembled dimers. We further identified the residues that affect the rigidity of the loop by mutagenesis and determined the structures of two PACSIN 3 wedge loop mutants. Our results suggest that the rigidity-mediated conformations of the wedge loop correlate well with the various crystal packing modes and membrane tubulations. Thus, the rigidity of the wedge loop is a key factor in dictating tubule diameters.

Identification of a novel nuclear-localized adenylate kinase from Drosophila melanogaster

As a step to further understand the role of adenylate kinase (AK) in the energy metabolism network, we identified, purified, and characterized a previously underscribed adenylate kinase in Drosophila melanogaster. The cDNA encodes a 175-amino acid protein, which shows 47.85% identity in 163 amino acids to human AK6. The recombinant protein was successfully expressed in Escherichia coli BL21 (DE3) strain. Characterization of this protein by enzyme activity assay showed adenylate kinase activity. AMP and CMP were the preferred substrates, and UMP can also be phosphorylated to some extent, with ATP as the best phosphate donor. Subcellular localization study showed a predominantly nuclear localization. Therefore, based on the substrate specificity, the specific nuclear localization in the cell, and the sequence similarity with human AK6, we named this novel adenylate kinase identified from the fly DAK6.

Crystallization and preliminary X-ray crystallographic analysis of human PACSIN 1 protein.

PACSIN 1, which is mainly detected in brain tissue, is one of the PACSIN-family proteins involved in endocytosis and recruitment of synaptic vesicles. It binds to dynamin, synaptojanin 1 and N-WASP, and functions in vesicle formation and transport. However, the mechanisms of action of PACSIN 1 in these processes are largely unknown. Here, full-length and five C-terminal truncation constructs of human PACSIN 1 have been successfully expressed and purified in Escherichia coli. PACSIN 1 (1-344) was crystallized and diffracted to a resolution of 3.0 A. The crystal belonged to space group C2, with unit-cell parameters a = 158.65, b = 87.38, c = 91.76 A, alpha = 90.00, beta = 113.61, gamma = 90.00 degrees . There were two molecules in the asymmetric unit and the solvent content was estimated to be about 70.47%.

Human secreted stabilin-1-interacting chitinase-like protein aggravates the inflammation associated with rheumatoid arthritis and is a potential macrophage inflammatory regulator in rodents.

To study the relationship between the human secreted protein stabilin‐1−interacting chitinase‐like protein (SI‐CLP) and rheumatoid arthritis (RA).

Crystallization and Preliminary X-Ray Crystallographic Studies on SICLP, a Novel Human Glyco_18 Domain – Containing Protein

A novel human Glyco_18 domain-containing protein, SI-CLP, was detected recently in human bronchoalveolar lavage of patients with chronic inflammatory disorders of the respiratory tract and peripheral-blood leukocytes. The expression of SI-CLP is up-regulated by dexamethasone or IL-4 and involved in the Th2 cell pathway. To further investigate its structure and function will provide new insights into human immunity and related disorders. Here we provide a preliminary crystal image of SI-CLP using the hanging-drop vapor diffusion method. The crystals of SI-CLP diffracted X-rays to a resolution of 2.7 A. The crystals belong to the space group P3(2)21 with unit cell parameters a=b=99.79 A, c=250.53 A, alpha=beta=90 degrees, gamma=120 degrees. There are two molecules per asymmetry unit.

Protein expression, crystallization and preliminary X-ray crystallographic studies of LidA from Legionella pneumophila

LidA, a translocated substrate of the Legionella pneumophila Dot/Icm type IV secretion system, is associated with maintenance of bacterial integrity and interferes with the early secretory pathway. However, the precise mechanism of LidA in these processes remains elusive. To further investigate the structure and function of LidA, the full-length protein was successfully expressed in Escherichia coli and purified. LidA was crystallized using sitting-drop vapour diffusion and diffracted to a resolution of 2.75 Å. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 57.5, b = 64.5, c = 167.3 Å, α = β = γ = 90°. There is one molecule per asymmetric unit.