Erika J. Mancini

Cryo-Electron Microscopy Reveals the Functional Organization of an Enveloped Virus, Semliki Forest Virus

Semliki Forest virus serves as a paradigm for membrane fusion and assembly. Our icosahedral reconstruction combined 5276 particle images from 48 cryo-electron micrographs and determined the virion structure to 9 A resolution. The improved resolution of this map reveals an N-terminal arm linking capsid subunits and defines the spike-capsid interaction sites. It illustrates the paired helical nature of the transmembrane segments and the elongated structures connecting them to the spike projecting domains. A 10 A diameter density in the fusion protein lines the cavity at the center of the spike. These clearly visible features combine with the variation in order between the layers to provide a framework for understanding the structural changes during the life cycle of an enveloped virus.

Atomic Snapshots of an RNA Packaging Motor Reveal Conformational Changes Linking ATP Hydrolysis to RNA Translocation

Many viruses package their genome into preformed capsids using packaging motors powered by the hydrolysis of ATP. The hexameric ATPase P4 of dsRNA bacteriophage phi12, located at the vertices of the icosahedral capsid, is such a packaging motor. We have captured crystallographic structures of P4 for all the key points along the catalytic pathway, including apo, substrate analog bound, and product bound. Substrate and product binding have been observed as both binary complexes and ternary complexes with divalent cations. These structures reveal large movements of the putative RNA binding loop, which are coupled with nucleotide binding and hydrolysis, indicating how ATP hydrolysis drives RNA translocation through cooperative conformational changes. Two distinct conformations of bound nucleotide triphosphate suggest how hydrolysis is activated by RNA binding. This provides a model for chemomechanical coupling for a prototype of the large family of hexameric helicases and oligonucleotide translocating enzymes.

Structure and functionality in flavivirus NS-proteins: perspectives for drug design.

Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.

A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results

An initial tranche of results from day-to-day use of a robotic system for setting up 100 nl-scale vapour-diffusion sitting-drop protein crystallizations has been surveyed. The database of over 50 unrelated samples represents a snapshot of projects currently at the stage of crystallization trials in Oxford research groups and as such encompasses a broad range of proteins. The results indicate that the nanolitre-scale methodology consistently identifies more crystallization conditions than traditional hand-pipetting-style methods; however, in a number of cases successful scale-up is then problematic. Crystals grown in the initial 100 nl-scale drops have in the majority of cases allowed useful characterization of x-ray diffraction, either in-house or at synchrotron beamlines. For a significant number of projects, full x-ray diffraction data sets have been collected to 3 A resolution or better (either in-house or at the synchrotron) from crystals grown at the 100 nl scale. To date, five structures have been determined by molecular replacement directly from such data and a further three from scale-up of conditions established at the nanolitre scale.

Crystal Structure of a Novel Conformational State of the Flavivirus NS3 Protein: Implications for Polyprotein Processing and Viral Replication

ABSTRACT The flavivirus genome comprises a single strand of positive-sense RNA, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. One of these, nonstructural protein 3 (NS3), is an enzyme with both serine protease and NTPase/helicase activities. NS3 plays a central role in the flavivirus life cycle: the NS3 N-terminal serine protease together with its essential cofactor NS2B is involved in the processing of the polyprotein, whereas the NS3 C-terminal NTPase/helicase is responsible for ATP-dependent RNA strand separation during replication. An unresolved question remains regarding why NS3 appears to encode two apparently disconnected functionalities within one protein. Here we report the 2.75-Å-resolution crystal structure of full-length Murray Valley encephalitis virus NS3 fused with the protease activation peptide of NS2B. The biochemical characterization of this construct suggests that the protease has little influence on the helicase activity and vice versa. This finding is in agreement with the structural data, revealing a single protein with two essentially segregated globular domains. Comparison of the structure with that of dengue virus type 4 NS2B-NS3 reveals a relative orientation of the two domains that is radically different between the two structures. Our analysis suggests that the relative domain-domain orientation in NS3 is highly variable and dictated by a flexible interdomain linker. The possible implications of this conformational flexibility for the function of NS3 are discussed.

Structure of the Leukemia Oncogene Lmo2: Implications for the Assembly of a Hematopoietic Transcription Factor Complex.

The LIM only protein 2 (LMO2) is a key regulator of hematopoietic stem cell development whose ectopic expression in T cells leads to the onset of acute lymphoblastic leukemia. Through its LIM domains, LMO2 is thought to function as the scaffold for a DNA-binding transcription regulator complex, including the basic helix-loop-helix proteins SCL/TAL1 and E47, the zinc finger protein GATA-1, and LIM-domain interacting protein LDB1. To understand the role of LMO2 in the formation of this complex and ultimately to dissect its function in normal and aberrant hematopoiesis, we solved the crystal structure of LMO2 in complex with the LID domain of LDB1 at 2.4 Å resolution. We observe a largely unstructured LMO2 kept in register by the LID binding both LIM domains. Comparison of independently determined crystal structures of LMO2 reveals large movements around a conserved hinge between the LIM domains. We demonstrate that such conformational flexibility is necessary for binding of LMO2 to its partner protein SCL/TAL1 in vitro and for the function of this complex in vivo. These results, together with molecular docking and analysis of evolutionarily conserved residues, yield the first structural model of the DNA-binding complex containing LMO2, LDB1, SCL/TAL1, and GATA-1.

Hexameric molecular motors: P4 packaging ATPase unravels the mechanism

Abstract.Genome packaging into an empty capsid is an essential step in the assembly of many complex viruses. In double-stranded RNA (dsRNA) bacteriophages of the Cystoviridae family this step is performed by a hexameric helicase P4 which is one of the simplest packaging motors found in nature. Biochemical and structural studies of P4 proteins have led to a surprising finding that these proteins bear mechanistic and structural similarities to a variety of the pervasive RecA/F1-ATPase-like motors that are involved in diverse biological functions. This review describes the role of P4 proteins in assembly, transcription and replication of dsRNA bacteriophages as it has emerged over the past decade while focusing on the most recent structural studies. The P4 mechanism is compared with the models proposed for the related hexameric motors.

High-resolution icosahedral reconstruction: fulfilling the promise of cryo-electron microscopy

Two recent papers have defined the secondary structure of the hepatitis virus capsid using a combination of cryo-electron microscopy and icosahedral image reconstruction. These two papers do more than reveal a new fold for a virus protein; they herald a new era in which image reconstruction of single particles will provide reliable high-resolution structural information. In revealing the promise of these techniques to the structural biology community, their two papers should play a seminal role for single particle work, similar to that of the work of Unwin and Henderson on bacteriorhodopsin in revealing the potential of electron microscopy of membrane protein crystals. Indeed, the success of these single particle methods owes much to the development of high-resolution techniques for two-dimensional crystals. This review will summarize some of the history of icosahedral reconstruction from cryo-electron micrographs, compare the two different approaches used to obtain the recent results and outline some of the challenges and promises for the future.

The Docking Interaction of Caspase-9 with ERK2 Provides a Mechanism for the Selective Inhibitory Phosphorylation of Caspase-9 at Threonine 125

Caspase-9 plays a critical role in the initiation of apoptosis by the mitochondrial pathway. Activation of caspase-9 is inhibited by phosphorylation at Thr125 by ERK1/2 MAPKs in response to growth factors. Here, we show that phosphorylation of this site is specific for these classical MAPKs and is not strongly induced when JNK and p38α/β MAPKs are activated by anisomycin. By deletion and mutagenic analysis, we identify domains in caspase-9 and ERK2 that mediate their interaction. Binding of ERK2 to caspase-9 and subsequent phosphorylation of caspase-9 requires a basic docking domain (D domain) in the N-terminal prodomain of the caspase. Mutational analysis of ERK2 reveals a 157TTCD160 motif required for recognition of caspase-9 that acts independently of the putative common docking domain. Molecular modeling supports the conclusion that Arg10 in the D domain of caspase-9 interacts with Asp160 in the TTCD motif of ERK2. Differences in the TTCD motif in other MAPK family members could account for the selective recognition of caspase-9 by ERK1/2. This selectivity may be important for the antiapoptotic role of classical MAPKs in contrast to the proapoptotic roles of stress-activated MAPKs.

Semi-automated microseeding of nanolitre crystallization experiments

A simple semi-automated microseeding procedure for nanolitre crystallization experiments is described. Firstly, a microseed stock solution is made from microcrystals using a Teflon bead. A dilution series of this microseed stock is then prepared and dispensed as 100 nl droplets into 96-well crystallization plates, facilitating the incorporation of seeding into high-throughput crystallization pipelines. This basic microseeding procedure has been modified to include additive-screening and cross-seeding methods. Five examples in which these techniques have been used successfully are described.