Viruses employ a variety of molecular strategies to carve out an existence in their host and to thwart host defenses. Crystal structures of viral proteins and of the host proteins deployed as ...molecular weapons contribute enormously to our understanding of viral pathogenesis and our efforts to combat viral infection. Mosquito‐borne flaviviruses, including the dengue, Zika, yellow fever, Japanese encephalitis and West Nile viruses, cause serious human diseases in much of the world. A growing number of diverse functions has been discovered for the enigmatic virulence factor known as NS1: it is an essential cofactor in viral genome replication, a mediator of the host immune response, and a trigger of host vascular leakage. NS1 lacks precedent in the structure and sequence databases, so an accurate 3D structure was key to understanding the basis for its various functions. A high‐throughput survey of expression conditions in baculovirus‐infected insect cells was essential to identifying conditions for production of recombinant NS1 in its natural glycosylated, disulfide‐linked form. We solved a crystal structure of West Nile virus NS1 from the anomalous scattering of the native sulfur atoms using an 18‐crystal data set 1,2. The structure revealed a previously unknown protein fold with a fundamentally dimeric architecture, and led to assigned functions for two of the three NS1 structural domains. Subsequent structures of NS1 from dengue virus and Zika viruses established the basis for its membrane association in cells and its lipid encapsidation when secreted 3. Recently discovered functions of this remarkable virulence factor emerged in follow‐up studies that built upon the crystal structures. Prime among these is the discovery that the NS1 protein, in a mouse model of dengue disease and in absence of virus, can induce the vascular leak that is a hallmark of severe dengue disease 4,5. Clues to the molecular mechanism of NS1‐induced endothelial dysfunction emerged from a study of the protection afforded by a monoclonal antibody and the structure of the Fab fragment bound to the NS1 epitope 6.
1 Akey DL et al. & Smith JL (2014) Science 343, 881‐885.
2 Akey DL et al. & Smith JL (2014) Acta Crystallogr D70, 2719‐2729.
3 Brown WC et al. & Smith JL (2016) Nature Struct Molec Biol 23, 856‐867.
4 Beatty PR et al. & Harris E (2015) Science Trans Med 7, 304ra141.
5 Modhiran N et al. & Young PR (2015) Science Trans Med 7, 304ra142.
6 Biering SB, Akey DL et al. Smith JL & Harris E (2021) Science 371, 194‐200.
DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to ...the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq). We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
This report presents the conclusions of the X-ray Validation Task Force of the worldwide Protein Data Bank (PDB). The PDB has expanded massively since current criteria for validation of deposited ...structures were adopted, allowing a much more sophisticated understanding of all the components of macromolecular crystals. The size of the PDB creates new opportunities to validate structures by comparison with the existing database, and the now-mandatory deposition of structure factors creates new opportunities to validate the underlying diffraction data. These developments highlighted the need for a new assessment of validation criteria. The Task Force recommends that a small set of validation data be presented in an easily understood format, relative to both the full PDB and the applicable resolution class, with greater detail available to interested users. Most importantly, we recommend that referees and editors judging the quality of structural experiments have access to a concise summary of well-established quality indicators.
► Validation criteria used by the PDB for X-ray crystal structures have been reassessed ► Key scores should be presented prominently in an easily understood format ► A concise validation report should be available to referees of papers on crystal structures
Micro-crystallography comes of age Smith, Janet L; Fischetti, Robert F; Yamamoto, Masaki
Current opinion in structural biology,
10/2012, Letnik:
22, Številka:
5
Journal Article
Recenzirano
Odprti dostop
► X-ray micro-beams permit structure determination from small or imperfect crystals. ► Robust micro-beams require source, optics and sample support to be extremely stable. ► Four different approaches ...to making a 1–20-μm beam have been implemented. ► A growing demand has led to ∼20 beamlines for micro-crystallography worldwide. ► Micro-beams enable data collection from LCP-grown crystals and crystals in situ.
The latest revolution in macromolecular crystallography was incited by the development of dedicated, user friendly, micro-crystallography beam lines. Brilliant X-ray beams of diameter 20μm or less, now available at most synchrotron sources, enable structure determination from samples that previously were inaccessible. Relative to traditional crystallography, crystals with one or more small dimensions have diffraction patterns with vastly improved signal-to-noise when recorded with an appropriately matched beam size. Structures can be solved from isolated, well diffracting regions within inhomogeneous samples. This review summarizes the technological requirements and approaches to producing micro-beams and how they continue to change the practice of crystallography.
DNA replication is essential for all living organisms. Several events can disrupt replication, including DNA damage (e.g., pyrimidine dimers, crosslinking) and so-called "roadblocks" (e.g., ...DNA-binding proteins or transcription). Bacteria have several well-characterized mechanisms for repairing damaged DNA and then restoring functional replication forks. However, little is known about the repair of stalled or arrested replication forks in the absence of chemical alterations to DNA. Using a library of random transposon insertions in Bacillus subtilis, we identified 35 genes that affect the ability of cells to survive exposure to an inhibitor that arrests replication elongation, but does not cause chemical alteration of the DNA. Genes identified include those involved in iron-sulfur homeostasis, cell envelope biogenesis, and DNA repair and recombination. In B. subtilis, and many bacteria, two nucleases (AddAB and RecJ) are involved in early steps in repairing replication forks arrested by chemical damage to DNA and loss of either nuclease causes increased sensitivity to DNA damaging agents. These nucleases resect DNA ends, leading to assembly of the recombinase RecA onto the single-stranded DNA. Notably, we found that disruption of recJ increased survival of cells following replication arrest, indicating that in the absence of chemical damage to DNA, RecJ is detrimental to survival. In contrast, and as expected, disruption of addA decreased survival of cells following replication arrest, indicating that AddA promotes survival. The different phenotypes of addA and recJ mutants appeared to be due to differences in assembly of RecA onto DNA. RecJ appeared to promote too much assembly of RecA filaments. Our results indicate that in the absence of chemical damage to DNA, RecA is dispensable for cells to survive replication arrest and that the stable RecA nucleofilaments favored by the RecJ pathway may lead to cell death by preventing proper processing of the arrested replication fork.
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Covering: up to the end of 2018
Polyketides are a valuable source of bioactive and clinically important molecules. The biosynthesis of these chemically complex molecules has led to the discovery of ...equally complex polyketide synthase (PKS) pathways. Crystallography has yielded snapshots of individual catalytic domains, di-domains, and multi-domains from a variety of PKS megasynthases, and cryo-EM studies have provided initial views of a PKS module in a series of defined biochemical states. Here, we review the structural and biochemical results that shed light on the protein-protein interactions critical to catalysis by PKS systems with an embedded acyltransferase. Interactions include those that occur both within and between PKS modules, as well as with accessory enzymes.
Protein-protein interactions of
cis
-AT polyketide synthases are dominated by the travels of the ACP domain to the active site entrance of each catalytic domain.
The Zika virus, which has been implicated in an increase in neonatal microcephaly and Guillain-Barré syndrome, has spread rapidly through tropical regions of the world. The virulence protein NS1 ...functions in genome replication and host immune-system modulation. Here, we report the crystal structure of full-length Zika virus NS1, revealing an elongated hydrophobic surface for membrane association and a polar surface that varies substantially among flaviviruses.
Infection of animal cells by numerous viruses is detected and countered by a variety of means, including recognition of nonself nucleic acids. The zinc finger antiviral protein (ZAP) depletes ...cytoplasmic RNA that is recognized as foreign in mammalian cells by virtue of its elevated CG dinucleotide content compared with endogenous mRNAs. Here, we determined a crystal structure of a protein-RNA complex containing the N-terminal, 4-zinc finger human (h) ZAP RNA-binding domain (RBD) and a CG dinucleotide-containing RNA target. The structure reveals in molecular detail how hZAP is able to bind selectively to CG-rich RNA. Specifically, the 4 zinc fingers create a basic patch on the hZAP RBD surface. The highly basic second zinc finger contains a pocket that selectively accommodates CG dinucleotide bases. Structure guided mutagenesis, cross-linking immunoprecipitation sequencing assays, and RNA affinity assays show that the structurally defined CG-binding pocket is not required for RNA binding per se in human cells. However, the pocket is a crucial determinant of high-affinity, specific binding to CG dinucleotide-containing RNA. Moreover, variations in RNA-binding specificity among a panel of CG-binding pocket mutants quantitatively predict their selective antiviral activity against a CG-enriched HIV-1 strain. Overall, the hZAP RBD RNA structure provides an atomic-level explanation for how ZAP selectively targets foreign, CG-rich RNA.
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