Janet L. Smith

A New Generation of Crystallographic Validation Tools for the Protein Data Bank

Summary 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.

Crystal structure of chloroplast cytochrome freveals a novel cytochrome fold and unexpected heme ligation

BACKGROUND Cytochrome f is the high potential electron acceptor of the chloroplast cytochrome b6f complex, and is the electron donor to plastocyanin. The 285-residue cytochrome f subunit is anchored in the thylakoid membrane of the chloroplast by a single membrane-spanning segment near the carboxyl terminus. A soluble redox-active 252-residue lumen-side polypeptide with native spectroscopic and redox properties, missing the membrane anchor and carboxyl terminus, was purified from turnip chloroplasts for structural studies. RESULTS The crystal structure of cytochrome f, determined to 2.3 A resolution, has several unexpected features. The 252-residue polypeptide is organized into one large and one small domain. The larger heme-binding domain is strikingly different from known structures of other c-type cytochromes and has the same fold as the type III domain of the animal protein, fibronectin. Cytochrome f binds heme with an unprecedented axial heme iron ligand: the amino terminus of the polypeptide. CONCLUSION The first atomic structure of a subunit of either the cytochrome b6f complex or of the related cytochrome bc1 complex has been obtained. The structure of cytochrome f allows prediction of the approximate docking site of plastocyanin and should allow systematic studies of the mechanism of intra- and inter-protein electron transfer between the cytochrome heme and plastocyanin copper, which are approximately isopotential. The unprecedented axial heme iron ligand also provides information on the sequence of events (i.e. cleavage of signal peptide and ligation of heme) associated with translocation of the cytochrome across the membrane and its subsequent folding.

Enzymes Utilizing Glutamine as an Amide Donor

Amide nitrogen from glutamine is a major source of nitrogen atoms incorporated biosynthetically into other amino acids, purine and pyrimidine bases, amino-sugars, and coenzymes. A family comprised of at least sixteen amidotransferases are known to catalyze amide nitrogen transfer from glutamine to their acceptor substrates. Recent fine structural advances, largely as a result of X-ray crystallography, now provide structure-based mechanisms that help to explain fundamental aspects of the catalytic and regulatory interactions of several of these aminotransferases. This chapter provides an overview of this recent progress made on the characterization of amidotransferase structure and mechanism.

The crystal structure of GMP synthetase reveals a novel catalytic triad and is a structural paradigm for two enzyme families.

The crystal structure of GMP synthetase serves as a prototype for two families of metabolic enzymes. The Class I glutamine amidotransferase domain of GMP synthetase is found in related enzymes of the purine, pyrimidine, tryptophan, arginine, histidine and folic acid biosynthetic pathways. This domain includes a conserved Cys-His-Glu triad and is representative of a new family of enzymes that use a catalytic triad for enzymatic hydrolysis. The structure and conserved sequence fingerprint of the nucleotide-binding site in a second domain of GMP synthetase are common to a family of ATP pyrophosphatases, including NAD synthetase, asparagine synthetase and argininosuccinate synthetase.

Flavivirus NS1 Structures Reveal Surfaces for Associations with Membranes and the Immune System

Two-Faced Viral Protein Flaviviruses cause human diseases such as West Nile fever and dengue fever. The flavivirus nonstructural protein 1 (NS1) has multiple functions in flavivirus biology and is a target for vaccine development. Dimeric NS1 is essential for replication of the viral genome inside host cells, while hexameric NS1 is secreted and plays a role in evasion of the immune system. Akey et al. (p. 881, published online 6 February; see the Perspective by Shi) report crystal structures for full-length glycosylated NS1 from West Nile and dengue viruses. The structures show a hexamer comprised of three dimers. The structural analysis together with liposome and mutational studies identify a membrane interacting surface on one face of the dimer and an immune evasion surface on the other. The structure of a viral protein provides a basis for understanding its function and could guide vaccine development. [Also see Perspective by Shi] Flaviviruses, the human pathogens responsible for dengue fever, West Nile fever, tick-borne encephalitis, and yellow fever, are endemic in tropical and temperate parts of the world. The flavivirus nonstructural protein 1 (NS1) functions in genome replication as an intracellular dimer and in immune system evasion as a secreted hexamer. We report crystal structures for full-length, glycosylated NS1 from West Nile and dengue viruses. The NS1 hexamer in crystal structures is similar to a solution hexamer visualized by single-particle electron microscopy. Recombinant NS1 binds to lipid bilayers and remodels large liposomes into lipoprotein nanoparticles. The NS1 structures reveal distinct domains for membrane association of the dimer and interactions with the immune system and are a basis for elucidating the molecular mechanism of NS1 function.

Structure of a modular polyketide synthase

Polyketide natural products constitute a broad class of compounds with diverse structural features and biological activities. Their biosynthetic machinery, represented by type I polyketide synthases (PKSs), has an architecture in which successive modules catalyse two-carbon linear extensions and keto-group processing reactions on intermediates covalently tethered to carrier domains. Here we used electron cryo-microscopy to determine sub-nanometre-resolution three-dimensional reconstructions of a full-length PKS module from the bacterium Streptomyces venezuelae that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intramodule carrier domain. In contrast, the carrier from the preceding module uses a separate entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time, to our knowledge, the structural basis for both intramodule and intermodule substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems.

Metamorphic enzyme assembly in polyketide diversification

Natural product chemical diversity is fuelled by the emergence and ongoing evolution of biosynthetic pathways in secondary metabolism. However, co-evolution of enzymes for metabolic diversification is not well understood, especially at the biochemical level. Here, two parallel assemblies with an extraordinarily high sequence identity from Lyngbya majuscula form a β-branched cyclopropane in the curacin A pathway (Cur), and a vinyl chloride group in the jamaicamide pathway (Jam). The components include a halogenase, a 3-hydroxy-3-methylglutaryl enzyme cassette for polyketide β-branching, and an enoyl reductase domain. The halogenase from CurA, and the dehydratases (ECH1s), decarboxylases (ECH2s) and enoyl reductase domains from both Cur and Jam, were assessed biochemically to determine the mechanisms of cyclopropane and vinyl chloride formation. Unexpectedly, the polyketide β-branching pathway was modified by introduction of a γ-chlorination step on (S)-3-hydroxy-3-methylglutaryl mediated by Cur halogenase, a non-haem Fe(ii), α-ketoglutarate-dependent enzyme. In a divergent scheme, Cur ECH2 was found to catalyse formation of the α,β enoyl thioester, whereas Jam ECH2 formed a vinyl chloride moiety by selectively generating the corresponding β,γ enoyl thioester of the 3-methyl-4-chloroglutaconyl decarboxylation product. Finally, the enoyl reductase domain of CurF specifically catalysed an unprecedented cyclopropanation on the chlorinated product of Cur ECH2 instead of the canonical α,β C = C saturation reaction. Thus, the combination of chlorination and polyketide β-branching, coupled with mechanistic diversification of ECH2 and enoyl reductase, leads to the formation of cyclopropane and vinyl chloride moieties. These results reveal a parallel interplay of evolutionary events in multienzyme systems leading to functional group diversity in secondary metabolites.

Structure of the Flavivirus Helicase: Implications for Catalytic Activity, Protein Interactions, and Proteolytic Processing

ABSTRACT Yellow fever virus (YFV), a member of the Flavivirus genus, has a plus-sense RNA genome encoding a single polyprotein. Viral protein NS3 includes a protease and a helicase that are essential to virus replication and to RNA capping. The 1.8-Å crystal structure of the helicase region of the YFV NS3 protein includes residues 187 to 623. Two familiar helicase domains bind nucleotide in a triphosphate pocket without base recognition, providing a site for nonspecific hydrolysis of nucleoside triphosphates and RNA triphosphate. The third, C-terminal domain has a unique structure and is proposed to function in RNA and protein recognition. The organization of the three domains indicates that cleavage of the viral polyprotein NS3-NS4A junction occurs in trans.

Micro-crystallography comes of age

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 20 μm 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.

Structural rearrangements of a polyketide synthase module during its catalytic cycle

The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents. The architecture of a full-length PKS module from the pikromycin pathway of Streptomyces venezuelae creates a reaction chamber for the intramodule acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase, ketosynthase and ketoreductase active sites (see accompanying paper). Here we determine electron cryo-microscopy structures of a full-length pikromycin PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry. The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of the ketosynthase, after extension to the β-keto intermediate, and after β-hydroxy product generation. The structures reveal the ACP dynamics for sequential interactions with catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. During the enzymatic cycle the ketoreductase domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the ACP-bound polyketide chain elongation intermediate. These findings have crucial implications for the design of functional PKS modules, and for the engineering of pathways to generate pharmacologically relevant molecules.