Max H. Nanao

Structural insight into cap-snatching and RNA synthesis by influenza polymerase.

Influenza virus polymerase uses a capped primer, derived by ‘cap-snatching’ from host pre-messenger RNA, to transcribe its RNA genome into mRNA and a stuttering mechanism to generate the poly(A) tail. By contrast, genome replication is unprimed and generates exact full-length copies of the template. Here we use crystal structures of bat influenza A and human influenza B polymerases (FluA and FluB), bound to the viral RNA promoter, to give mechanistic insight into these distinct processes. In the FluA structure, a loop analogous to the priming loop of flavivirus polymerases suggests that influenza could initiate unprimed template replication by a similar mechanism. Comparing the FluA and FluB structures suggests that cap-snatching involves in situ rotation of the PB2 cap-binding domain to direct the capped primer first towards the endonuclease and then into the polymerase active site. The polymerase probably undergoes considerable conformational changes to convert the observed pre-initiation state into the active initiation and elongation states.

Structure of the Dengue Virus Helicase/Nucleoside Triphosphatase Catalytic Domain at a Resolution of 2.4 Å

ABSTRACT Dengue fever is an important emerging public health concern, with several million viral infections occurring annually, for which no effective therapy currently exists. The NS3 protein from Dengue virus is a multifunctional protein of 69 kDa, endowed with protease, helicase, and nucleoside 5′-triphosphatase (NTPase) activities. Thus, NS3 plays an important role in viral replication and represents a very interesting target for the development of specific antiviral inhibitors. We present the structure of an enzymatically active fragment of the Dengue virus NTPase/helicase catalytic domain to 2.4 Å resolution. The structure is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Its C-terminal domain adopts a new fold compared to the NS3 helicase of hepatitis C virus, which has interesting implications for the evolution of the Flaviviridae replication complex. A bound sulfate ion reveals residues involved in the metal-dependent NTPase catalytic mechanism. Comparison with the NS3 hepatitis C virus helicase complexed to single-stranded DNA would place the 3′ single-stranded tail of a nucleic acid duplex in the tunnel that runs across the basic face of the protein. A possible model for the unwinding mechanism is proposed.

Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression

Significance Auxin is a critical plant hormone that regulates every aspect of plant growth and development. AUXIN RESPONSE FACTOR (ARF) transcription factors control auxin-regulated gene transcription, and their activity is regulated by AUXIN/INDOLE 3-ACETIC ACID repressor proteins. This work identifies that dimerization of the repressor with the transcription factor is insufficient to repress activity, suggesting that multimerization is the mechanism of repressing ARF transcriptional activity and further raising the possibility that multimerization in other systems may play roles in transcriptional repression. In plants, the AUXIN RESPONSE FACTOR (ARF) transcription factor family regulates gene expression in response to auxin. In the absence of auxin, ARF transcription factors are repressed by interaction with AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) proteins. Although the C termini of ARF and Aux/IAA proteins facilitate their homo- and heterooligomerization, the molecular basis for this interaction remained undefined. The crystal structure of the C-terminal interaction domain of Arabidopsis ARF7 reveals a Phox and Bem1p (PB1) domain that provides both positive and negative electrostatic interfaces for directional protein interaction. Mutation of interface residues in the ARF7 PB1 domain yields monomeric protein and abolishes interaction with both itself and IAA17. Expression of a stabilized Aux/IAA protein (i.e., IAA16) bearing PB1 mutations in Arabidopsis suggests a multimerization requirement for ARF protein repression, leading to a refined auxin-signaling model.

Crystal Structure and Mechanism of Activation of TANK-Binding Kinase 1

Tank-binding kinase I (TBK1) plays a key role in the innate immune system by integrating signals from pattern-recognition receptors. Here, we report the X-ray crystal structures of inhibitor-bound inactive and active TBK1 determined to 2.6 Å and 4.0 Å resolution, respectively. The structures reveal a compact dimer made up of trimodular subunits containing an N-terminal kinase domain (KD), a ubiquitin-like domain (ULD), and an α-helical scaffold dimerization domain (SDD). Activation rearranges the KD into an active conformation while maintaining the overall dimer conformation. Low-resolution SAXS studies reveal that the missing C-terminal domain (CTD) extends away from the main body of the kinase dimer. Mutants that interfere with TBK1 dimerization show significantly reduced trans-autophosphorylation but retain the ability to bind adaptor proteins through the CTD. Our results provide detailed insights into the architecture of TBK1 and the molecular mechanism of activation.

Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins

Plant Hormone Modulators The activity and stability of several plant hormones is modulated by conjugation with various amino acids and their derivatives. Westfall et al. (p. 1708, published online 24 May) solved the crystal structures for two acyl acid amido synthetases from Arabidopsis. The findings suggest how the enzymes might discriminate between apolar and acidic amino acids and lend insight into the reaction chemistries that add functional diversity to hormone signaling pathways. Crystal structures of plant GH3 proteins reveal how these enzymes accommodate jasmonates, auxins, and benzoates. Acyl acid amido synthetases of the GH3 family act as critical prereceptor modulators of plant hormone action; however, the molecular basis for their hormone selectivity is unclear. Here, we report the crystal structures of benzoate-specific Arabidopsis thaliana AtGH3.12/PBS3 and jasmonic acid–specific AtGH3.11/JAR1. These structures, combined with biochemical analysis, define features for the conjugation of amino acids to diverse acyl acid substrates and highlight the importance of conformational changes in the carboxyl-terminal domain for catalysis. We also identify residues forming the acyl acid binding site across the GH3 family and residues critical for amino acid recognition. Our results demonstrate how a highly adaptable three-dimensional scaffold is used for the evolution of promiscuous activity across an enzyme family for modulation of plant signaling molecules.

Structural basis for oligomerization of auxin transcriptional regulators

The plant hormone auxin is a key morphogenetic regulator acting from embryogenesis onwards. Transcriptional events in response to auxin are mediated by the auxin response factor (ARF) transcription factors and the Aux/IAA (IAA) transcriptional repressors. At low auxin concentrations, IAA repressors associate with ARF proteins and recruit corepressors that prevent auxin-induced gene expression. At higher auxin concentrations, IAAs are degraded and ARFs become free to regulate auxin-responsive genes. The interaction between ARFs and IAAs is thus central to auxin signalling and occurs through the highly conserved domain III/IV present in both types of proteins. Here, we report the crystal structure of ARF5 domain III/IV and reveal the molecular determinants of ARF-IAA interactions. We further provide evidence that ARFs have the potential to oligomerize, a property that could be important for gene regulation in response to auxin.

Crystal structure of human otubain 2

Ubiquitylation, the modification of cellular proteins by the covalent attachment of ubiquitin, is critical for diverse biological processes including cell cycle progression, signal transduction and stress response. This process can be reversed and regulated by a group of proteases called deubiquitylating enzymes (DUBs). Otubains are a recently identified family of DUBs that belong to the ovarian tumour (OTU) superfamily of proteins. Here, we report the first crystal structure of an OTU superfamily protein, otubain 2, at 2.1 Å resolution and propose a model for otubain–ubiquitin binding on the basis of other DUB structures. Although otubain 2 is a member of the cysteine protease superfamily of folds, its crystal structure shows a novel fold for DUBs. Moreover, the active‐site cleft is sterically occluded by a novel loop conformation resulting in an oxyanion hole, which consists uniquely of backbone amides, rather than the composite backbone/side‐chain substructures seen in other DUBs and cysteine proteases. Furthermore, the residues that orient and stabilize the active‐site histidine of otubain 2 are different from other cysteine proteases. This reorganization of the active‐site topology provides a possible explanation for the low turnover and substrate specificity of the otubains.

ISPyB: an information management system for synchrotron macromolecular crystallography

MOTIVATION Individual research groups now analyze thousands of samples per year at synchrotron macromolecular crystallography (MX) resources. The efficient management of experimental data is thus essential if the best possible experiments are to be performed and the best possible data used in downstream processes in structure determination pipelines. Information System for Protein crystallography Beamlines (ISPyB), a Laboratory Information Management System (LIMS) with an underlying data model allowing for the integration of analyses down-stream of the data collection experiment was developed to facilitate such data management. RESULTS ISPyB is now a multisite, generic LIMS for synchrotron-based MX experiments. Its initial functionality has been enhanced to include improved sample tracking and reporting of experimental protocols, the direct ranking of the diffraction characteristics of individual samples and the archiving of raw data and results from ancillary experiments and post-experiment data processing protocols. This latter feature paves the way for ISPyB to play a central role in future macromolecular structure solution pipelines and validates the application of the approach used in ISPyB to other experimental techniques, such as biological solution Small Angle X-ray Scattering and spectroscopy, which have similar sample tracking and data handling requirements.

Automatic processing of macromolecular crystallography X-ray diffraction data at the ESRF

A system for the automatic reduction of single- and multi-position macromolecular crystallography data is presented.

Phasing in the presence of radiation damage

In the accurate estimation of small signals, redundancy of observations is often seen as an essential tool for the experimenter. This is particularly true during macromolecular structure determination by single-wavelength anomalous dispersion (SAD), where the exploitable signal can be less than a few percent. At the most intense undulator synchrotron beamlines, the effect of radiation damage can be such that all usable signal is obscured. Here the magnitude of this effect in experiments performed at the Se K-edge is quantified. Six successive data sets were collected on the same crystal, interspersed with two exposures to the X-ray beam during which data were not collected. It is shown that the very first data set has excellent phasing statistics, whereas these statistics degrade for the later data sets. Merging several data sets into one, highly redundant, data set only gave moderate improvements as a result of the presence of radiation damage. Part of the damage could be corrected for using a linear interpolation scheme. Interpolation of the data to a low-dose as well as to a high-dose data set allowed us to combine the SAD method with the radiation-damage induced phasing (RIP) technique, which further improved the experimental phases, especially after density modification. Some recommendations are given on how to mitigate the effect of radiation damage during structure determination.