ABSTRACT
Spt5 (NusG in bacteria) is the only RNA polymerase-associated factor known to be conserved in all three domains of life. In archaea and eukaryotes, Spt5 associates with Spt4, an elongation factor that is absent in bacteria, to form a functional heterodimeric complex. Previous studies suggest that the Spt4:Spt5 complex interacts directly with DNA at the double-stranded DNA exit tunnel of RNA polymerase to regulate gene transcription. In this study, the DNA-binding ability of Spt4:Spt5 from the archaeon Methanocaldococcus jannaschii was confirmed via nuclear magnetic resonance chemical shift perturbation and fluorescence polarization assays. Crystallographic analysis of the full-length MjSpt4:Spt5 revealed two distinct conformations of the C-terminal KOW domain of Spt5. A similar alkaline region was found on the Spt4:Spt5 surface in both crystal forms, and identified as double-stranded DNA binding patch through mutagenesis-fluorescence polarization assays. Based on these structural and biochemical data, the Spt4:Spt5-DNA binding model was built for the first time.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Methanocaldococcus/genetics , Transcriptional Elongation Factors/metabolism , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Electrophoretic Mobility Shift Assay , Models, Molecular , Multiprotein Complexes/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, TertiaryABSTRACT
SUCROSE-NON-FERMENTING1-RELATED PROTEIN KINASE1 (SnRK1), a central plant metabolic sensor kinase, phosphorylates its target proteins, triggering a global shift from anabolism to catabolism. Molecular modeling revealed that upon binding of KIN10 to GEMINIVIRUS REP-INTERACTING KINASE1 (GRIK1), KIN10's activation T-loop reorients into GRIK1's active site, enabling its phosphorylation and activation. Trehalose 6-phosphate (T6P) is a proxy for cellular sugar status and a potent inhibitor of SnRK1. T6P binds to KIN10, a SnRK1 catalytic subunit, weakening its affinity for GRIK1. Here, we investigate the molecular details of T6P inhibition of KIN10. Molecular dynamics simulations and in vitro phosphorylation assays identified and validated the T6P binding site on KIN10. Under high-sugar conditions, T6P binds to KIN10, blocking the reorientation of its activation loop and preventing its phosphorylation and activation by GRIK1. Under these conditions, SnRK1 maintains only basal activity levels, minimizing phosphorylation of its target proteins, thereby facilitating a general shift from catabolism to anabolism.
Subject(s)
Arabidopsis Proteins , Protein Serine-Threonine Kinases , Sugar Phosphates , Trehalose , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/antagonists & inhibitors , Arabidopsis Proteins/chemistry , Binding Sites , Molecular Dynamics Simulation , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Sugar Phosphates/metabolism , Transcription Factors , Trehalose/analogs & derivatives , Trehalose/metabolismABSTRACT
Alkane monooxygenase (AlkB) is a widely occurring integral membrane metalloenzyme that catalyzes the initial step in the functionalization of recalcitrant alkanes with high terminal selectivity. AlkB enables diverse microorganisms to use alkanes as their sole carbon and energy source. Here we present the 48.6-kDa cryo-electron microscopy structure of a natural fusion from Fontimonas thermophila between AlkB and its electron donor AlkG at 2.76 Å resolution. The AlkB portion contains six transmembrane helices with an alkane entry tunnel within its transmembrane domain. A dodecane substrate is oriented by hydrophobic tunnel-lining residues to present a terminal C-H bond toward a diiron active site. AlkG, an [Fe-4S] rubredoxin, docks via electrostatic interactions and sequentially transfers electrons to the diiron center. The archetypal structural complex presented reveals the basis for terminal C-H selectivity and functionalization within this broadly distributed evolutionary class of enzymes.
Subject(s)
Alkanes , Cryoelectron Microscopy , Cytochrome P-450 CYP4A/chemistry , Alkanes/chemistryABSTRACT
Through a structure-based irreversible drug design approach, we have discovered a highly potent IDH1-mutant inhibitor compound 16 (IHMT-IDH1-053) (IC50 = 4.7 nM), which displays high selectivity against IDH1 mutants over IDH1 wt and IDH2 wt/mutants. The crystal structure demonstrates that 16 binds to the IDH1 R132H protein in the allosteric pocket adjacent to the NAPDH binding pocket through a covalent bond with residue Cys269. 16 inhibits 2-hydroxyglutarate (2-HG) production in IDH1 R132H mutant transfected 293T cells (IC50 = 28 nM). In addition, it inhibits the proliferation of HT1080 cell line and primary AML cells which both bear IDH1 R132 mutants. In vivo, 16 inhibits 2-HG level in a HT1080 xenograft mouse model. Our study suggested that 16 would be a new pharmacological tool to study IDH1 mutant-related pathology and the covalent binding mode provided a novel approach for designing irreversible IDH1 inhibitors.
Subject(s)
Enzyme Inhibitors , Isocitrate Dehydrogenase , Mice , Humans , Animals , Isocitrate Dehydrogenase/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Cell Line , Drug Design , MutationABSTRACT
The recent developments at microdiffraction X-ray beamlines are making microcrystals of macromolecules appealing subjects for routine structural analysis. Microcrystal diffraction data collected at synchrotron microdiffraction beamlines may be radiation damaged with incomplete data per microcrystal and with unit-cell variations. A multi-stage data assembly method has previously been designed for microcrystal synchrotron crystallography. Here the strategy has been implemented as a Python program for microcrystal data assembly (PyMDA). PyMDA optimizes microcrystal data quality including weak anomalous signals through iterative crystal and frame rejections. Beyond microcrystals, PyMDA may be applicable for assembling data sets from larger crystals for improved data quality.
ABSTRACT
HER2, a member of the epidermal growth factor receptor (EGFR) family, has been associated with human breast, ovarian and gastric cancers. Anti-HER2 monoclonal antibodies (mAbs) have demonstrated clinical efficacy for HER2-overexpressing breast cancer. A chimeric antibody chA21 that specifically inhibits the growth of HER2-overexpressing cancer cells both in vitro and in vivo has previously been developed. To reduce a potential human anti-mouse immune response, the humanized antibody HuA21 was developed and was further subjected to affinity maturation by phage display on the basis of chA21. Here, the crystal structure of HuA21-scFv in complex with the extracellular domain of HER2 is reported, which demonstrates that HuA21 binds almost the same epitope as chA21 and also provides insight into how substitutions in HuA21 improve the binding affinity compared with chA21, which could facilitate structure-based optimization in the future. Furthermore, the effects of HuA21 variants with constant domains of different lengths were explored and it was noticed that the deletion of constant domain 1 could improve the inhibition efficacy in a cell-proliferation assay, possibly functioning via increased internalization, which might guide the design of other monoclonal antibodies.
Subject(s)
Antibodies, Monoclonal, Humanized/chemistry , Antigen-Antibody Complex/chemistry , Antineoplastic Agents, Immunological/chemistry , Neoplasms/therapy , Receptor, ErbB-2/antagonists & inhibitors , Animals , Antibodies, Monoclonal, Humanized/genetics , Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Agents, Immunological/therapeutic use , Binding Sites, Antibody , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Crystallization , Crystallography, X-Ray/methods , Female , Humans , Protein Domains , Receptor, ErbB-2/immunologyABSTRACT
De novo structural evaluation of native biomolecules from single-wavelength anomalous diffraction (SAD) is a challenge because of the weakness of the anomalous scattering. The anomalous scattering from relevant native elements - primarily sulfur in proteins and phospho-rus in nucleic acids - increases as the X-ray energy decreases toward their K-edge transitions. Thus, measurements at a lowered X-ray energy are promising for making native SAD routine and robust. For microcrystals with sizes less than 10â µm, native-SAD phasing at synchrotron microdiffraction beamlines is even more challenging because of difficulties in sample manipulation, diffraction data collection and data analysis. Native-SAD analysis from microcrystals by using X-ray free-electron lasers has been demonstrated but has required use of thousands of thousands of microcrystals to achieve the necessary accuracy. Here it is shown that by exploitation of anomalous microdiffraction signals obtained at 5â keV, by the use of polyimide wellmounts, and by an iterative crystal and frame-rejection method, microcrystal native-SAD phasing is possible from as few as about 1 200 crystals. Our results show the utility of low-energy native-SAD phasing with microcrystals at synchrotron microdiffraction beamlines.
ABSTRACT
The anti-apoptotic transmembrane Bax inhibitor motif (TMBIM) containing protein family regulates Ca2+ homeostasis, cell death, and the progression of diseases including cancers. The recent crystal structures of the TMBIM homolog BsYetJ reveal a conserved Asp171-Asp195 dyad that is proposed in regulating a pH-dependent Ca2+ translocation. Here we show that BsYetJ mediates Ca2+ fluxes in permeabilized mammalian cells, and its interaction with Ca2+ is sensitive to protons and other cations. We report crystal structures of BsYetJ in additional states, revealing the flexibility of the dyad in a closed state and a pore-opening mechanism. Functional studies show that the dyad is responsible for both Ca2+ affinity and pH dependence. Computational simulations suggest that protonation of Asp171 weakens its interaction with Arg60, leading to an open state. Our integrated analysis provides insights into the regulation of the BsYetJ Ca2+ channel that may inform understanding of human TMBIM proteins regarding their roles in cell death and diseases.
Subject(s)
Calcium/metabolism , Membrane Proteins/metabolism , Protons , Amino Acid Motifs , Apoptosis , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Crystallography, X-Ray , HeLa Cells , Humans , Hydrogen-Ion Concentration , Membrane Proteins/chemistry , Membrane Proteins/genetics , Molecular Dynamics Simulation , Protein BindingABSTRACT
With the recent developments in microcrystal handling, synchrotron microdiffraction beamline instrumentation and data analysis, microcrystal crystallo-graphy with crystal sizes of less than 10â µm is appealing at synchrotrons. However, challenges remain in sample manipulation and data assembly for robust microcrystal synchrotron crystallography. Here, the development of micro-sized polyimide well-mounts for the manipulation of microcrystals of a few micrometres in size and the implementation of a robust data-analysis method for the assembly of rotational microdiffraction data sets from many microcrystals are described. The method demonstrates that microcrystals may be routinely utilized for the acquisition and assembly of complete data sets from synchrotron microdiffraction beamlines.
ABSTRACT
The Gos1 protein (Golgi SNAP receptor complex member 1) is involved in the SNARE complex, which is the core machinery that drives membrane fusion between cargo-carrying vesicles and their target membranes in the secretory and endocytic pathways in yeast. Truncated versions of the Gos1 protein from Saccharomyces cerevisiae were cloned, expressed, purified and crystallized. The crystal belonged to space group P212121, with unit-cell parameters a=39.67, b=43.58, c=81.94â Å, α=ß=γ=90°. An X-ray diffraction data set was collected at 100â K to 1.63â Å resolution. Matthews coefficient (VM) calculations suggest that one molecule is present in the asymmetric unit, corresponding to a solvent content of â¼55%.